Identification of MicroRNAs Involved in Different Layers of rice-Magnaporthe oryzae interaction

Identification of MicroRNAs Involved in Different Layers of rice-Magnaporthe oryzae interaction | 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 Identification of MicroRNAs Involved in Different Layers of rice-Magnaporthe oryzae interaction Sadam Hussain Bhutto, Yong Zhu, Hao Su, Hao Xue, Xiao-Yu Xiong, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7070474/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2025 Read the published version in Rice → Version 1 posted 10 You are reading this latest preprint version Abstract Rice blast disease is one of the most destructive rice diseases worldwide. Rice MicroRNAs (miRNAs) play an essential role in immunity against blast fungus Magnaporthe oryzae . However, it remains unclear which miRNAs are involved in the three layers of rice- M. oryzae interaction, including pathogen associated molecular patterns (PAMPs)-triggered immunity (PTI), effector-triggered susceptibility (ETS), and effector-triggered immunity (ETI). In this study, we performed small RNA-sequencing to systemically identify miRNAs regulating PTI, ETS, and ETI in rice- M. oryzae interaction. A totally 441 miRNAs were identified, with 13, 30, and 14 miRNAs screened out and classified as regulators of PTI, ETS, and ETI, respectively. We investigated and confirmed the roles of 9 previously reported miRNAs and an uncharacterized miRNA, miR408-5p, in the three interaction processes. We demonstrated that miR1320-5p positively regulated PTI; miR396 family members and miR164a improved, whereas miR171b and miR172a suppressed ETS; miR166a enhanced, whereas miR169a and miR396 family members suppressed ETI. Moreover, we demonstrated that miR397b and miR408-5p enhanced rice susceptibility by promoting ETS and suppressing ETI; miR398b enhanced rice resistance by promoting both PTI and ETI while suppressing ETS. Our findings figured a miRNA-mediated regulatory network in which distinct miRNAs modulate PTI, ETS, and ETI against M. oryzae . This study provides theoretical support and genetic resources for disease-resistant breeding in rice. rice blast disease miRNAs Small RNA-sequencing PTI ETS ETI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The plant-microbe interaction contains three-layers. The first layer is known as PTI, which is triggered by the recognition of the microbe-derived PAMPs by plant pattern recognition receptors (PRRs). Successful pathogens deploy effectors to facilitate pathogen virulence and interfere with PTI, leading to the second layer interaction, namely effector-triggered susceptibility (ETS). In turn, the recognition of the effectors by the cognate resistance ( R ) genes leads to the third layer interaction, i.e. effector-triggered immunity, which mostly rely on immune receptor genes encoding nucleotide-binding site leucine-rich repeat (NLR) proteins(Jones and Dangl, 2006 ). Increasing evidence indicates that these three-layered plant-pathogen interactions are fine-tuned by a subset of microRNAs. MicroRNAs (miRNAs) are a class of 20–24 nucleotides (nt) non-coding RNAs that suppress the expression of genes containing complementary sequences (Yu et al., 2017 ). In plants, miRNAs play crucial roles in fine-tuning growth, development, and immunity (Yu et al., 2017 ; Li et al., 2019 ). Currently, increasing evidence supported the involvement of miRNAs in regulating both PTI and ETI responses. miR393 is the first miRNA identified to be involving in plant immunity. In Arabidopsis, miR393 modulates PTI and ETI responses against Pseudomonas syringae pv. tomato (Pst) DC3000 by targeting the auxin receptor genes TIR1 , AFB2 , and AFB3 . The accumulation of miR393 is induced by the bacterial PAMP flg22, thereby enhancing basal defence responses (Navarro et al., 2006 ). However, bacterial effectors, such as AvrPto and AvrPtoB, suppresses miR393 accumulation, interfering with PTI (Navarro et al., 2008 ). Similarly, miR160a accumulation is induced by flg22, a well-characterized PAMP derived from bacterial flagellin that triggers PTI (Li et al., 2010 ). In contrast, miR472 regulates ETI by silencing R genes such as RPS5 , which activates ETI against P. syringe by recognizing the cognate effector AvrPphB (Boccara et al., 2014 ). In soybean, miR393 and miR166 accumulate during Phytophthora sojae infection to contribute to PTI (Wong et al., 2014 ). Additionally, several miRNAs, such as miR1510a and miR2109 in soybean, miR482 and miR2118 in tomato, all directly target resistance R genes (Zhai et al., 2011 ; Shivaprasad et al., 2012 ; Yan et al., 2022 ). Furthermore, in barley, miR9863 family members target NLRs , the Mla alleles, thereby dampening immune responses triggered by MLAs (Liu et al., 2014 ). Rice ( Oryza sativa ) is one of the most important crops supporting half of the world population. However, rice blast, caused by fungal pathogen Magnaporthe oryzae , is the most destructive rice disease worldwide, leading to 10–30% yield loss yearly (Devanna et al., 2022 ). In rice, PTI and ETI play crucial roles in defence against M. oryzae (Chen and Ronald, 2011 ). For instance, PRRs such as Chitin Elicitor Receptor Kinase 1 (CERK1), Chitin Elicitor Binding protein (CEBiP), Lysin Motif–Containing Protein 4 (LYP4), and LYP6 recognize PAMP chitin and mediate PTI (Shimizu et al., 2010 ; Liu et al., 2012 ). The R gene Piz-t recognizes M. oryzae -derived effector AvrPiz-t to activate ETI (Li et al., 2009 ). In turn, when there are no cognate R genes in rice, the effectors are virulent and trigger ETS. For instance, in rice accessions lacking Piz-t , AvrPiz-t acts as a virulent factor by suppressing the ubiquitin ligase activity of AvrPiz-t Interacting Protein 6 (APIP6) thereby repressing basal defence responses and enhancing susceptibility to M. oryzae (Park et al., 2012 ). In contrast, M. oryzae mutants defective in effector secretion exhibit severely attenuated virulence, even losing the ability to establish successful infection. For example, the M. oryzae gene ELO1 encodes a fatty acid elongase, which is a rate-limiting enzyme in very-long-chain fatty acid (VLCFA) biosynthesis (He et al., 2020 ). Disruption of MoELO1 specifically blocks appressorium-mediated plant infection due to severely impaired penetration peg formation and invasive hypha expansion (He et al., 2020 ), ultimately preventing effector delivery into rice cells. To date, over 700 mature miRNAs have been identified in rice genome, with approximately 70 of them being responsive to M. oryzae , suggesting their involvement in rice immunity (Li et al., 2014 ; Baldrich et al., 2015 ). Among these miRNAs, quite a few have been identified as regulators of rice blast disease resistance. For example, 16 miRNAs act as negative regulators of blast resistance, including miR156, miR164a, miR167d, miR168a, miR169a, miR319, miR396, miR439, miR444b, miR530, miR535, miR1432, miR1871, miR1873, miR1875, and miR9664 (Feng et al., 2021 ) (Li et al., 2021 ; Li et al., 2021 ; Li et al., 2021 ; Lu et al., 2021 ; Wang et al., 2021 ; Li et al., 2022 ; Zhang et al., 2022 ; Sheng et al., 2023 ). In contrast, nine miRNAs have been characterized as positive regulators, including, miR159a, miR160a, miR162, miR166h-66k, miR171b, miR172a, miR398b, miR812w, and miR7695(Salvador-Guirao et al., 2018 ; Li et al., 2019 ; Li et al., 2020 ; Chen et al., 2021 ; Sánchez-Martín and Keller, 2021 ; Feng et al., 2022 ; Li et al., 2022 ; Wang et al., 2025 ). However, it is unclear whether and which miRNAs are specifically involved in PTI, ETS, and ETI in rice – M . oryzae interactions. In this study, we performed small RNA sequencing (small RNA-seq) under distinct experimental conditions to identify the miRNAs that may specifically act in PTI, ETS, and ETI in rice. We identified the miRNAs involved in PTI by comparing the susceptible line Lijiangxin Tuan Heigu (LTH) with or without chitin treatment, the miRNAs involved in ETS by analysing LTH infected with the virulence M. oryzae strain Guy11 or the avirulent strain Δ Moelo (defective in appressorium formation and invasive hyphal growth), and the miRNAs involved in ETI by examine a transgenic line expressing R gene Piz-t inoculated with a virulence strain RB22 or an incompatible strain carrying AvrPiz-t (RB22(AvrPiz-t)). We also examined the blast disease resistance, PTI responses, and ETI-related hypersensitive responses in the rice transgenic lines overexpressing miRNAs or by transiently expressing these miRNAs in Nicotiana benthamiana . Our results demonstrated that distinct miRNAs modulate PTI, ETS, and ETI during rice- M. oryzae interactions. Results Deep-Sequencing Analysis of Small RNA Libraries To identify miRNAs involved in rice PTI, we spray-inoculated three-leaf-stage LTH seedlings with chitin or water (mock control). We collected the inoculated leaves at 0,3-, and 6-hours post-inoculation (hpi) with three biological replicates per time point for RNA extraction and constructed 15 small RNA libraries for small RNA high-throughput sequencing. The rice genome-matched miRNA reads ranged from 148,048 to 328,635 in 14 libraries except for a 3-hour chitin-treated sample with matched reads 57,178 (Table S1 ). To identify miRNAs involved in rice ETS, we spray-inoculated three-leaf-stage LTH seedlings with either a virulent M. oryzae strain Guy11 or an avirulent strain Δ Moelo . Δ Moelo is a mutant strain with deletion of the Moelo gene, which is defective in both penetration peg formation and invasive hyphal growth(He et al., 2020 ). We hypothesized that ΔMoelo fails to secret effectors into rice cells, thus significantly attenuating ETS. Therefore, the miRNAs showing differential responses to Guy11 and Δ Moelo are specifically involved in ETS. LTH displayed visible disease lesions after Guy11 infection but showed invisible symptoms upon ΔMoelo inoculation (Fig. 1 A). The infected leaves were collected at 0, 12, and 24 hpi with three biological replicates per time points for RNA extraction. We constructed 15 small RNA libraries for high-throughput sequencing, which yielded rice genome-matched miRNA reads ranging from 47,851 to 271,369 across all libraries (Table S1 ). To identify miRNAs involved in rice ETI, we spray-inoculated the three-leaf-stage seedlings of a transgenic line expressing the R gene Piz-t in Nipponbare background (NPB ( Piz-t:HA ) with either a compatible M. oryzae strain RB22 or an incompatible RB22 strain carrying AvrPiz-t, which would trigger ETI after recognition by its cognate R gene Piz-t (Li et al., 2009 ; Park et al., 2012 ). We speculated that miRNAs involved in ETI should be differentially responsive to the two strains. As expected, Piz-t:HA seedlings showed disease symptoms upon RB22 infection, whereas displayed no symptom upon RB22 (AvrPiz-t) inoculation (Fig. 1 B). The infected leaves were collected at 0, 18, and 42 hpi with three biological replicates for RNA extraction. We constructed 15 small RNA libraries for high-throughput sequencing. The rice genome-matched miRNA reads ranged from 121,727 to 285,367 in 15 libraries (Table S1 ). To evaluate the consistency of experimental replicates across different treatments, we performed principal component analysis (PCA) to cluster the samples in different treatment groups. The PCA results showed that the samples from the same treatment group exhibited comparable patterns and were clustered together. All PTI-related miRNAs were placed in Group 1, ETS-related in Group 2, and ETI-related in Group 3 (Fig. S1 A), indicating a high consistency among the biological replicates. We further assessed sample similarity by analysing sample-to-sample distances based on miRNA profiles. The distance heatmap revealed distinct miRNA accumulation patterns among treatment (Fig. S1 B). The size distribution analysis showed that the majority of small RNAs in our libraries were 19–24 nt (Fig. 1 C), consistent with the characteristic sizes of miRNAs and siRNAs {Mi, 2008 #343}. We identified 441 known miRNAs in these libraries (Table S2), covering most of the known rice miRNAs and validating the reliability of our sequencing data. Identification of miRNAs Acting in Rice PTI The miRNAs involved in PTI were expected to be responsive to chitin in a universally susceptible rice accession LTH. We compared the miRNA reads in chitin-treated and mock samples of LTH using screening criteria including a p- value < 0.05, Fold change ˃ 2 or 5. In comparison with the mock samples, no miRNAs were detected at 3-hour post-treatment (hpt), while 13 miRNAs were identified at 6 hpt, including 10 upregulated and three down-regulated (Fig. 2 A, Table S3). To validate our sequencing results, we randomly selected six miRNAs and examined their expression pattern by stem-loop real-time PCR analysis in LTH. The expression patterns of these miRNAs were basically consistent with the sequencing results: chitin induced miR1320-5p, miR398b, miR2887, and miR5814, whereas suppressed miR166g-5p and miR396e-5p (Fig. 2 B). Moreover, Gene Ontology (GO) analysis of predicted miRNA target genes showed significant enrichment in biological process of “Response to the biotic stimulus” (Fig. S2A), implying that these miRNAs play potential roles in PTI by regulating their target genes. Among these miRNAs, miR1320 and miR398b have been previously characterized as positive regulators of rice blast disease resistance (Li et al., 2019 ; Wang et al., 2021 ). In contrast, the members of miR396 family, such as miR396a, miR396b, miR396d, and miR396h, have been identified as negative regulator (Chandran et al., 2019 ). We then examined PAMP-triggered defence responses, including the reactive oxygen species (ROS) burst and the induction of defence-related genes, in transgenic lines overexpressing miR1320 ( OX1320 ). Compared to the Kasalath control, the OX1320 lines displayed enhanced ROS accumulation and increased defence-related gene-expression levels following flg22 and chitin treatments (Fig. 2 C-G), suggesting that miR1320 enhances PTI in rice. Since miR396e-5p target the same genes as the other miR396 family members mentioned above ( https://www.zhaolab.org/psRNATarget/ ), we analysed PTI-related ROS burst in transgenic lines overexpressing miR396 family members ( OX396a , OX396c , OX396a , and OX396c ). However, the OX396 lines displayed a similar or slightly elevated ROS accumulation compared to the TP309 control (Fig. S3A-B), indicating that the tested miR396 members may not play a significant role in PTI and different members have different functionally preferentiality as reported in our previous study (Chandran et al., 2019 ). Identification of miRNAs Acting in Rice ETS The miRNAs involved in ETS should be deferentially responsive to Guy11 and ΔMoelo in LTH that has PTI. To identify ETS-related miRNAs, we compared miRNA reads between Guy11-infected and ΔMoelo- infected LTH samples using the following screening criteria including a p- value < 0.05, Fold change ˃ 2 or 5 across all libraries. Compared to ΔMoelo -infected samples that have PTI to prevent infection, 23 miRNAs were upregulated and 7 miRNAs were down-regulated in Guy11-infected samples at 12 and 24 hpi (Fig. 3 A, Table S4). To validate these finding, we randomly selected 10 miRNAs and examined their expression patterns. Compared to ΔMoelo , Guy11 infection promoted the accumulation of miR398b, miR171b, miR164d, and miR396e at both 12- and 24-hours post-inoculation (hpi), miR397b and miR408-5p at 24 hpi, whereas suppressed the accumulation of miR399d at 12 hpi, miR1862e, miR2877, and miR5814 at 24 hpi (Fig. 3 B). These expression patterns were consistent with the sequencing data, confirming their reliability. Moreover, GO analysis revealed that the target genes of these ETS-related miRNAs were also clustered into biological process of “Response to the biotic stimulus” (Fig. S2B), implying that these miRNAs were involved in ETS via their target genes. Among these upregulated miRNAs, members of the miR164 family have been characterized as negative regulators of rice blast disease resistance (Wang et al., 2018 ). Similarly, members of the miR396 family have also been identified as negative regulators (Chandran et al., 2019 ). These finding suggest that both miRNA families may enhance ETS, thereby compromising rice blast disease resistance. In contrast, miR171b and miR172a, which have been characterized as positive regulators, were both upregulated by Guy11 (Li et al., 2022 ; Wang et al., 2025 ). To determine whether these miRNAs regulate ETS, we assessed the resistance of the transgenic lines overexpressing either these miRNAs or their family members when challenged with the virulent isolate Guy11 and avirulent strain ΔMoelo . Upon infection with the virulent strains, both OX171b and OX172a transgenic lines exhibited enhanced disease resistance against virulent Guy11, with fewer disease lesions and less fungal growth compared to the Nipponbare control after punch-inoculation; in contrast, these lines supported the infection of ΔMoelo mutant strain (Fig. 3 C-F). Conversely, OX396 lines exhibited enhanced susceptibility to Guy11, with larger disease lesions and more fungal growth than the Nipponbare control, while maintaining similar symptom and fungal growth levels upon ΔMoelo inoculation (Fig. 3 G-H). Although OX164a exhibited increased sensitivity to both Guy11 and ΔMoelo , the fold change in fungal biomass for ΔMoelo was significantly lower than that for Guy11 (Fig. 3 I-J). Collectively, these findings indicate that miR171b and miR172a enhances rice blast disease resistance by suppressing ETS, whereas miR396 and miR164a improve susceptibility by enhancing ETS. Identification of miRNAs Acting in Rice ETI The miRNAs involved in ETI should be deferentially responsive to RB22 and RB22 (AvrPiz-t) in Piz-t-expressed plants. We compared miRNA expression profiles between RB22-infected and RB22 (AvrPiz-t)-infected Piz-t:HA samples, identifying differentially expressed miRNAs using screening criteria including a p- value < 0.05, Fold change ˃ 2 or 5 across all libraries. Compared to RB22-infected samples, 7 miRNAs were upregulated and 7 were downregulated in RB22 (AvrPiz-t)-infected samples at 18 or 42 hpi (Fig. 4 A, Table S5). To validate these results, we randomly selected 7 miRNAs and examined their expression patterns. Compared to the virulent isolate RB22, the incompatible strain RB22 (AvrPiz-t) upregulated the expression of miR166j-3p, miR398b, and miR1882e-3p at 18 and 42 hpi, and miR1862e at 18 hpi, whereas suppressed miR396a-5p, miR397b, and miR408-5p at 18 hpi (Fig. 4 B). These results are closely matched with the sequencing data, confirming their reliability. GO analysis showed that the target genes of these ETI-related miRNAs were enriched in the biological process “Secondary metabolic process” (Fig. S2C), a category tightly associated with defence responses, implying that these miRNAs may participate in ETI by regulating their target genes. Among these upregulated miRNAs, miR166j is from a miRNA family with members being characterized as a positive regulator of rice blast disease resistance (Salvador-Guirao et al., 2018 ), suggesting its positive regulatory role in ETI. In contrast, miR169 family members were known as negative regulators of blast disease resistance (Li et al., 2017 ). miR169e shows high sequence similarity to miR169a, differing by two nucleotides, one at the 5’ end and one at the 3’ ends, targeting the same genes. Therefore, we used the transgenic lines overexpressing miR169a ( OX169a ) for subsequent experiments. To investigate whether these miRNAs participate in ETI, we examined their effects on ETI-related cell-death by transiently co-expressing them in N. benthamiana along with the rice R genes Pik1 and Pik2 and their cognate effector Avr-PikD . Avr-PikC , which is not recognized by Pik , served as a negative control (De la Concepcion et al., 2018 ). miR166j accelerated Avr-PikD -triggered cell death and enhanced ion leakage, whereas miR396c, and miR169a delayed cell death and reduced ion leakage (Fig. 4 C-D). Next, we examined the roles of these miRNAs in ETI induced by incompatible M. oryzae isolates. FJ81278 is an incompatible isolate to Nipponbare derived from Fujian province, the south of China (Yang, 2021 ). We found that this isolate is also incompatible to TP309 and Kasalath (Fig. S4). Transgenic lines overexpressing miR166 were sterile, thus resistance could not be evaluated. Both OX169a and OX396 lines exhibited compatible interactions characterized by larger lesions and more fungal biomass compared to their controls upon FJ81278 infection (Fig. 4 E-H). Consistently, these transgenic lines also displayed enhanced susceptibility to compatible strain GZ8 (Fig. 4 E-H). These results indicated that miR169 and miR396 enhance rice susceptibility to M. oryzae by suppressing ETI. Both miR397b and miR408b-5p Enhance ETS While Suppress ETI We next identified miRNAs involved in two or three layers of rice – M. oryzae interactions. Venn analysis revealed that four miRNAs, including miR2877, miR396e-5p, miR398b, and miR5814, common to both PTI and ETS (Fig. S3C). OX396 lines displayed comparable or increased ROS accumulation relative to the TP309 control (Fig. S3A-B), suggesting that miR396e likely functions in ETS suppressing PTI. Five miRNAs, namely miR397b, miR398b, miR408-5p, miR1862e, and miR1882e-3p, were common to both ETS and ETI (Fig. 5 A). We generated transgenic lines overexpressing miR397b ( OX397b ) and miR408-5p ( OX408 ) in Nipponbare background, respectively (Fig. 5 B-C). The three-leaf-stage seedlings were inoculated with M. oryzae virulent strain GZ8, avirulent strain ΔMoelo , and incompatible isolates FJ81278. Both OX397b and OX40 8 lines showed enhanced susceptibility to GZ8 and FJ81278 with bigger disease lesions and more fungal biomass than Nipponbare control, whereas exhibited similar incompatible lesions indifferent from the control (Fig. 5 D-E). We further investigated their roles in effector-induced HR. When transiently co-expressed with Piks and their cognate effector Avr-PikD , both miRNAs greatly delayed cell death and significantly suppressed HR-triggered ion leakage (Fig. 5 F-H). collectively, these results indicate that miR397b and miR408-5p likely enhances rice susceptibility by promoting ETS and suppressing ETI. miR398b Participates in PTI, ETS, and ETI Venn analysis revealed that miR398b was common to all PTI, ETS, and ETS (Fig. 6 A). We previously have characterized it as a positive regulator of blast resistance and PTI {Li, 2019 #162}. However, its roles in ETS and ETI remained unexplored. We then analysed the roles of miR398b in PAMP-induced ROS burst and ETI-related HR, as well as the resistance to virulent and incompatible strains. The transgenic lines overexpressing miR398b ( OX398b ) showed higher and more ROS accumulation (Fig. 6 B). OX398b displayed enhanced resistance to the virulent strain NC-10 and GZ8, as well as the incompatible isolate FJ81278 with smaller disease lesions and less fungal biomass than the Kasalath control (Fig. 6 C-D), while similar response to the avirulent strain ΔMoelo . Consistently, miR398b greatly delayed cell death and significantly suppressed HR-triggered ion leakage (Fig. 6 E-F). Taken together, these results indicated that miR398b enhanced rice blast disease resistance by improving both PTI and ETI, presumably via suppressing ETS. Discussion Here we screened out 48 miRNAs specifically in PTI, ETS, and/or ETI (Fig. 7 ). Among these miRNAs, 12 miRNAs, including miR164, miR166, miR168, miR169, miR171, miR172, miR396, miR397b, miR398b, miR444, miR1320-5p, and miR1432-5p, have been previously characterized as regulators of rice blast disease resistance. We functionally characterized nine of these known plus one previously uncharacterized miRNA, miR408-5p, across all the three layers of rice – M. oryzae interaction. Our results indicate that these miRNAs orchestrate rice blast resistance through distinct mechanisms. miR1320 enhances resistance via regulating PTI; miR171b and miR172a enhances, whereas miR164 and miR396 families suppresses resistance via regulating ETS; miR166 enhances, whereas miR169 and miR396 suppress resistance via regulating ETI. Moreover, miR397b and miR408-5p suppress resistance via enhancing ETS while suppressing ETI; miR398b enhances resistance via improving PTI and ETI, while suppressing ETS. Our results reveal a complicated regulatory network that miRNAs collaboratively modulate multi-layered defence responses, thereby regulating blast disease resistance. Among the miRNAs identified as PTI regulators through deep sequencing, miR1432 have been characterized as a negative regulator of PTI in rice. Compared to the Nipponbare control, the transgenic lines overexpressing miR1432 showed compromised ROS burst and callose deposition induced by PAMPs, whereas the transgenic lines overexpressing its target gene EF-hand family protein 1 ( EFH1 ) exhibited enhanced defence responses (Li et al., 2021 ). In this study, deep sequencing data showed that chitin treatment enhances the accumulation of miR1432 (Fig. 2 A), confirming that miR1432 facilitated rice susceptibility via suppressing PTI. However, the mechanism by which miR1432-EFH1 regulatory module acts in PTI needs further investigation. In this study, we identified several miRNAs by deep sequencing, whose family members were previously characterized. Notably, miR396e-5p was identified as a regulator of both PTI and ETS. In a previous study, we have characterized the roles of another four miR396 family members in rice blast disease resistance, including miR396a-5p, miR396c-5p, miR396d-5p, and miR396h-5p(Chandran et al., 2019 ). Although these family members and miR396e-5p exhibited 1–2 nucleotide variations, they maintain functional conservation through target identical genes ( https://www.zhaolab.org/psRNATarget/ ). OX396 lines showed comparable ROS burst similar to the Nipponbare control (Fig. S3A-B), indicating that miR396 likely does not modulate PTI. Conversely, all the overexpressing lines of the four family members exhibited enhanced ETS phenotypes (Fig. 3 G-H), indicating that miR396 members likely facilitate rice susceptibility by improving ETS. In rice, miR396 targets 12 growth-regulating factors (GRFs) that orchestrate diverse physiological processes, such as grain size determination, inflorescence development, plant architecture determination, blast disease resistance, salt tolerance, and brown planthopper resistance (Liu et al., 2014 ; Gao et al., 2016 ; Li et al., 2018 ; Chandran et al., 2019 ; Dai et al., 2019 ; Chen et al., 2020 ; Chen et al., 2020 , 2024 ). However, it is worth further investigation of the downstream signalling pathways of miR396-GRFs module in ETS. Another identified miRNA, miR169e, differs from miR169a by two-nucleotides variation, while maintain functional conservation through target identical genes nuclear transcription factor Y-A family members ( NF-YAs ) ( https://www.zhaolab.org/psRNATarget/ ). Therefore, we hypothesized that miR169e and miR169a share functional conservation in regulating rice blast resistance. We previously demonstrated that miR169a negatively regulates blast disease resistance (Li et al., 2017 ), although its molecular mechanism remained unclear. In this study, we found that miR169a overexpressing lines exhibited enhanced susceptibility to both compatible and incompatible strains (Fig. 4 E-F), suggesting that miR169 family members facilitate rice susceptibility by suppressing ETI. Notably, NF-YAs suppress jasmonic acid (JA)-mediated antiviral defence and promote susceptibility to rice stripe virus (RSV) and Southern rice black-streaked dwarf virus (SRBSDV) (Tan et al., 2022 ). Whether miR169- NF-YAs module regulates ETI in rice via JA signalling is unknown and need further study. In this study, we identified and characterized miR408-5p as novel regulator of both ETS and ETI. miR408 is a highly conserved miRNA family in plant widely modulating stress response, growth, and developments. In wheat, Tae-miR408 suppresses wheat responses to high-salinity, heavy cupric stress, and stripe rust by targeting a gene encoding a chemocyanin-like protein (Feng et al., 2013 ). In maize, miR408b negatively regulates resistance against Fusarium verticilliodes , the agent of maize Fusarium ear rot (FER) (Zhou et al., 2020 ). Here we showed that OX408 lines exhibited susceptibility to the incompatible strain and enhanced susceptibility to virulent strain compared to the Nipponbare control (Fig. 5 D-E), suggesting that miR408 likely enhances rice susceptibility by suppressing ETI. However, OX408 lines showed no significant difference in symptom development when challenged with Δ Moelo , which impaired in appressorium formation and invasive hypha development (both required for effector secretion), indicating that miR408 likely does not participate in PTI. Among the 48 miRNAs identified in this study, the function of 36 miRNAs in rice blast disease resistance remain uncharacterized. Notably, miR1862e emerged as a particularly promising candidate based on high accumulation in rice and identification in both ETS and ETI, suggesting its potential importance in rice- M. oryzae interactions. However, the roles of miR1862e were not ever been reported. Given this finding, future research should prioritize functional characterization of miR1862e in disease resistance and related molecular mechanism. Conclusion In this study, we identified miRNAs involved in PTI, ETS, and ETI during the rice– M. oryzae interaction and selected a total of 48 miRNAs for further analysis. We functionally characterized the roles of 10 of these miRNAs across all three layers of immunity. Our results reveal a complex regulatory network in which miRNAs collaboratively modulate multi-layered defence responses, thereby regulating blast disease resistance. These findings provide theoretical support and genetic resources for disease-resistant rice breeding. Experimental Procedures Growth Condition and Plant Material The rice ( Oryzae sativa ) plants were used in this study, including Japonica accessions Lijiangxin Tuan Heigu (LTH), Nipponbare, Piz-t lines that expressing HA-fused R gene Piz-t in Nipponbare background ( Piz-t:HA ), Indica accessions TP309, Kasalath, and the transgenic lines overexpressing miRNAs in indicated background. These rice plants were grown in a plant growth chamber under the controlled conditions with a temperature of 25 ± at 2°C Day/night, 70% relative humidity and a 14/10-h light/dark period. Pathogen Infection M. oryzae strains or isolates were used for blast infection, including Guy11, GZ8, NC-10, FJ81278, Δ Moelo , RB22, and RB22 expressing an M. oryzae effector ( AvrPiz-t ). These strains were grown on oat-tomato agar medium for 2 weeks at 28°C with a 12/12-h day/night treatment for sporulation. After getting rid of the surface mycelia with distilled water, the plates were further incubated for 3 days with consistent light treatment to promote sporulation. Spores were collected with concentration 5×10 5 conidia mL − 1 . The five-leaf-stage seedlings were spray inoculated for small RNA library construction or punch-inoculated for resistance assay. For punch inoculation, the leaves were slightly wounded with a mouse-ear punch, and the conidia suspension was punch-inoculated on wounded sites. The disease symptoms were observed at 5–7 days post-inoculation (dpi). The relative fungal biomass on infected leaves was measured according to a previous report (Park et al., 2012 ). The infected leaves were collected for DNA extraction. The relative fungal biomass was determined using the DNA amount of fungal MoPot2 against rice DNA amount of ubiquitin (UBQ) through q-PCR. Library Construction, Sequencing, and Bioinformatics Analysis of Small RNAs The small RNA library construction and Illumina sequencing were conducted following a previous report with some revision(Mi et al., 2008 ). In a summary, the total RNA extracted from the different treatment samples were used for small RNA library construction and RNA-sequencing (Oebiotech Company). Each treatment contains three repeats. The small RNA reads with length over 16 were mapped to the rice genomes ( http://rice.plantbiology.msu.edu/ ; version 7.0). The perfect genome-matched small RNAs were analysed and the normalized abundance of small RNAs was calculated as reads per million. Plasmid Construction and Genetic Transformation To construct the transgenic lines overexpressing miR397b and miR408-5p, the sequence on the MIR397b gene containing 278 bp upstream to 281 bp downstream and MIR804-5p gene containing 275 bp upstream to 276 bp downstream sequence was amplified from NPB genomic DNA with primers miR397b-F, miR397b-R and miR408-5p-F, and miR408-5p-R (Supplementary Table S10). PCR products were cloned into the binary vector 35S-pCAMBIA1300. The constructs were transformed into Nipponbare via Agrobacterium tumefaciens- mediated transformation. Transgenic plants were screened by PCR with the forward primer 35S-F and the specific reverse primers miR397b-R and miR408-5p-R (Supplemental Table S10). The positive transgenic lines were used for resistance analysis. RNA Extraction and Gene Expression Analysis For defence-related gene expression assay, three-leaf-stage rice seedlings of indicated lines were spray-inoculated with chitin or flg22, and the samples were collected at indicated time points. For miRNA transgenic line identification, the leaves from the T0 transgenic lines were collected. Total RNAs were extracted from the collected leaves using TRIzol reagent (No. 7E2582B5, Vazyme, NanJing, China). To determine the expression of PTI-related genes, the first-strand cDNA was synthesized from 1 µg of total RNA using PrimeScript RT Reagent Kit with gDNA Eraser following the manufacturer’s instructions (RR047A, TaKaRa). RT-qPCR was performed using specific primers and SYBR Green mix (QuantiNova SYBR Green PCR Kit; Qiagen) with the BIO-RAD C1000TM Thermal Cycler (Bio-Rad Inc., China). The rice ubiquitin ( UBQ ) gene was used as an internal reference to normalize the relative expression levels of genes. To examine the amount of miR408 and miR397b, total RNA was reverse-transcribed using a miRNA-specific stem-loop RT primer (Table S10), and the RT product was subsequently used as a template for RT-qPCR by using miRNA-specific forward primers and the universal reverse primer (Table S10). snRNA U6 was used as an internal reference to normalize the relative amounts of miRNAs. RT-qPCR analyses were performed with three technical replicates. Reactive Oxygen Species (ROS) The leaves of five-leaf-stage rice seedling were selected for ROS burst assay. The leaves were cut into 5mm-width squares with 1-mm-width stripes and incubated in 200 µL water in a 96-well plate for 12–16 h. Then, the squares were treated with or without 20 µg/mL chitin or 1 µM flg22 in 200 µL buffer containing 10 µg/mL horseradish peroxidase (Sigma-Aldrich, Shanghai, China) and 20 mM L-012 (Wako, Japan). The production of ROS was detected using a GLOMAX96 Microplate Luminometer (Promega Biotech Co., Ltd, Beijing, China) for 30–60 min and determined as relative luminescence units. Hypersensitive Response (HR) and Conductivity To investigate the cell death regulated by miRNAs, the cDNA sequences of Pik1 and Pik2 were amplified from ZH11 cDNA using indicated primers (Supplementary Table S10). Avr-PikC and Avr-PikD cDNA sequences were amplified from Guy11 cDNA using indicated primers (Supplementary Table S10). The amplified PCR products were cloned into the Kpn 1 and Sac 1 restriction sites of the pCAMBIA1300 vector and transformed into Agrobacterium tumefaciens GV3101. These strains were grown in lysogeny broth (LB) liquid media at 28 ºC overnight and resuspended in buffer (10 mM 2-(N-morpholino) ethanesulfonic acid [MES] [pH 5.5–5.7], 10 mM MgCl2, 200 mM acetosyringone) and injected into Nicotiana benthamiana leaves for transient expression. The phenotype was observed and the photo were capture with UV-light after 4–5 dpi. Then, the infected leaves were cut with a holler cutter and the leaf dices were transferred into the 10 ml tubes with sterilized ddH 2 O and kept at room temperature overnight. Then the tubes were boiled at 100°C for 5 minutes and kept for cooling at room temperature for 2 hours. After cooling, the conductance was measured with the help of a conductivity meter. Abbreviations PAMP pathogen-associated molecular pattern PTI PAMP-triggered immunity ETS effector-triggered susceptibility ETI effector-triggered immunity ROS Reactive oxygen species HR Hypersensitive response RT-qPCR Real-time quantitative polymerase chain reaction LTH Lijiang xin Tuan Heigu NPB Nipponbare TP309 Taipei309 GRFs Growth-regulating factors NF-YAs Nuclear transcription factor Y-A family members Declarations Ethical Approval and Consent to participate Not applicable Consent for publication Not applicable Availability of data and material The data and material mentioned in this study is available upon requirement. Competing Interests The authors declare no conflicts of interests. Funding This work was supported by the National Natural Science Foundation of China (No. 32121003, 32172417, 32372553, and 32401845), Science and technology department of Sichuan province (2025ZNSFSC1115, SCCXTD-2024-SD-4, and 2021YFYZ0021-4-2), and the Open Research Fund of State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China (SKL-ZD202202, SKL-ZY202202, SKL-ZY202205, and SKL-ZY202209). Authors’ Contributions W-M. Wang and Y. Li designed the experiments. S-H. Bhutto, Y. Z., H. S., X. H., X-Y. X., and Y. Yang conducted the experiments. Y-H. Z., H-S D., S-Y. Z., and D-Q. Li conducted the field trials. X-M. Y., H. W., G-B L., Z-X. Z., and J-W. Zhang analysed the data. W-M. W. and Y. Li wrote the paper. Y-Y. H. and M-I. Khaskheli discussed the results and commented on the manuscript. Acknowledgements We are grateful to Prof. Guo-Liang Wang at Ohio State University for providing RB22 and RB22(AvrPiz-t) strains, as well as the Piz-t:HA transgenic lines. 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Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACPmYIzcPAwHyAIQHCMcCrhQ2hhS2BSC0IJg9cJQEt7DyGnwsY7snwt/d8/vCgxi6xgb15mwRDzR08DuMxlp7BUMwjcebsNomEY8mJDTzHyiQYjj3Dp8VAmochgcdAIncbQ2IDc2KDRI6ZBGPDYby2/AZrkX/z+ENiQ31ig/wbglrMoLbwMEgkNhwG2sJDSAtbmTVIi8SZNDOgX44bt/GkFVskHMOthZ//8ObbQC32/O2HH3/8UVMt289+eOONDzW4tTAwcBgwMP5DthdEJODRwMDA/gCv9CgYBaNgFIwCBgD6u0Ti/S2JbAAAAABJRU5ErkJggg==","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-08 04:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7070474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7070474/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12284-025-00855-8","type":"published","date":"2025-10-24T16:16:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86967975,"identity":"5e6ed9cb-1dfa-494f-a237-d5ad0f18be5c","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":845944,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/d55eebe63ac660d38c89b50d.jpg"},{"id":86967974,"identity":"2f39e20a-64ee-4d79-9ce1-2a73aeb89eaf","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1349414,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/762269c48d382d4e0784029c.jpg"},{"id":86967977,"identity":"029858fa-586c-4d7a-a0bc-2110641d05a8","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1035133,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/f3c5b015bf3544fe1826ec2a.jpg"},{"id":86968404,"identity":"41ddd365-f095-4c6a-8dae-425d8e90f40d","added_by":"auto","created_at":"2025-07-17 18:08:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1086796,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/39341593e0d36fd040e63bd8.jpg"},{"id":86967982,"identity":"537f00d0-4958-4a24-8d7c-8038d050c388","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1313879,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/42a468f4d3f7888f1dd7dc97.jpg"},{"id":86967983,"identity":"238adfeb-3284-47e1-8c2a-664eb5dedf85","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1252074,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/b0b5cf01f596e911a5f0dc3f.jpg"},{"id":86967987,"identity":"2a791324-618d-41e2-8758-4a3c5ff7b82e","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":705531,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"FiguresandSupfigures7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/0abf593dd4803da0fcf81ad6.jpg"},{"id":94490466,"identity":"042cc6ce-5238-40bc-b34e-2eb7d3b3ff08","added_by":"auto","created_at":"2025-10-27 17:10:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8480737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/5ca4ec23-9505-43ae-8158-61d157caa344.pdf"},{"id":86968405,"identity":"6b132529-5fa6-4985-8484-50d2f0fb414c","added_by":"auto","created_at":"2025-07-17 18:08:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":259253,"visible":true,"origin":"","legend":"","description":"","filename":"SupTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/5b6b660c3433d6cc71129933.xlsx"},{"id":86967980,"identity":"3dd2dcec-0a71-4f7f-8f16-54cf4d21a47a","added_by":"auto","created_at":"2025-07-17 18:00:37","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6778932,"visible":true,"origin":"","legend":"","description":"","filename":"Supfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7070474/v1/0639b1088ddb933424e4bb54.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of MicroRNAs Involved in Different Layers of rice-Magnaporthe oryzae interaction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe plant-microbe interaction contains three-layers. The first layer is known as PTI, which is triggered by the recognition of the microbe-derived PAMPs by plant pattern recognition receptors (PRRs). Successful pathogens deploy effectors to facilitate pathogen virulence and interfere with PTI, leading to the second layer interaction, namely effector-triggered susceptibility (ETS). In turn, the recognition of the effectors by the cognate resistance (\u003cem\u003eR\u003c/em\u003e) genes leads to the third layer interaction, i.e. effector-triggered immunity, which mostly rely on immune receptor genes encoding nucleotide-binding site leucine-rich repeat (NLR) proteins(Jones and Dangl, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIncreasing evidence indicates that these three-layered plant-pathogen interactions are fine-tuned by a subset of microRNAs. MicroRNAs (miRNAs) are a class of 20\u0026ndash;24 nucleotides (nt) non-coding RNAs that suppress the expression of genes containing complementary sequences (Yu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In plants, miRNAs play crucial roles in fine-tuning growth, development, and immunity (Yu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Currently, increasing evidence supported the involvement of miRNAs in regulating both PTI and ETI responses. miR393 is the first miRNA identified to be involving in plant immunity. In Arabidopsis, miR393 modulates PTI and ETI responses against \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. tomato (Pst) \u003cem\u003eDC3000\u003c/em\u003e by targeting the auxin receptor genes \u003cem\u003eTIR1\u003c/em\u003e, \u003cem\u003eAFB2\u003c/em\u003e, and \u003cem\u003eAFB3\u003c/em\u003e. The accumulation of miR393 is induced by the bacterial PAMP flg22, thereby enhancing basal defence responses (Navarro et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, bacterial effectors, such as AvrPto and AvrPtoB, suppresses miR393 accumulation, interfering with PTI (Navarro et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, miR160a accumulation is induced by flg22, a well-characterized PAMP derived from bacterial flagellin that triggers PTI (Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In contrast, miR472 regulates ETI by silencing \u003cem\u003eR\u003c/em\u003e genes such as \u003cem\u003eRPS5\u003c/em\u003e, which activates ETI against \u003cem\u003eP. syringe\u003c/em\u003e by recognizing the cognate effector AvrPphB (Boccara et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In soybean, miR393 and miR166 accumulate during \u003cem\u003ePhytophthora sojae\u003c/em\u003e infection to contribute to PTI (Wong et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, several miRNAs, such as miR1510a and miR2109 in soybean, miR482 and miR2118 in tomato, all directly target resistance \u003cem\u003eR\u003c/em\u003e genes (Zhai et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Shivaprasad et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, in barley, miR9863 family members target \u003cem\u003eNLRs\u003c/em\u003e, the \u003cem\u003eMla\u003c/em\u003e alleles, thereby dampening immune responses triggered by \u003cem\u003eMLAs\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e) is one of the most important crops supporting half of the world population. However, rice blast, caused by fungal pathogen \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e, is the most destructive rice disease worldwide, leading to 10\u0026ndash;30% yield loss yearly (Devanna et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In rice, PTI and ETI play crucial roles in defence against \u003cem\u003eM. oryzae\u003c/em\u003e(Chen and Ronald, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For instance, PRRs such as Chitin Elicitor Receptor Kinase 1 (CERK1), Chitin Elicitor Binding protein (CEBiP), Lysin Motif\u0026ndash;Containing Protein 4 (LYP4), and LYP6 recognize PAMP chitin and mediate PTI (Shimizu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003eR\u003c/em\u003e gene \u003cem\u003ePiz-t\u003c/em\u003e recognizes \u003cem\u003eM. oryzae\u003c/em\u003e-derived effector AvrPiz-t to activate ETI (Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In turn, when there are no cognate \u003cem\u003eR\u003c/em\u003e genes in rice, the effectors are virulent and trigger ETS. For instance, in rice accessions lacking \u003cem\u003ePiz-t\u003c/em\u003e, AvrPiz-t acts as a virulent factor by suppressing the ubiquitin ligase activity of AvrPiz-t Interacting Protein 6 (APIP6) thereby repressing basal defence responses and enhancing susceptibility to \u003cem\u003eM. oryzae\u003c/em\u003e (Park et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In contrast, \u003cem\u003eM. oryzae\u003c/em\u003e mutants defective in effector secretion exhibit severely attenuated virulence, even losing the ability to establish successful infection. For example, the \u003cem\u003eM. oryzae\u003c/em\u003e gene \u003cem\u003eELO1\u003c/em\u003e encodes a fatty acid elongase, which is a rate-limiting enzyme in very-long-chain fatty acid (VLCFA) biosynthesis (He et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Disruption of \u003cem\u003eMoELO1\u003c/em\u003e specifically blocks appressorium-mediated plant infection due to severely impaired penetration peg formation and invasive hypha expansion (He et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), ultimately preventing effector delivery into rice cells.\u003c/p\u003e\u003cp\u003eTo date, over 700 mature miRNAs have been identified in rice genome, with approximately 70 of them being responsive to \u003cem\u003eM. oryzae\u003c/em\u003e, suggesting their involvement in rice immunity (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Baldrich et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among these miRNAs, quite a few have been identified as regulators of rice blast disease resistance. For example, 16 miRNAs act as negative regulators of blast resistance, including miR156, miR164a, miR167d, miR168a, miR169a, miR319, miR396, miR439, miR444b, miR530, miR535, miR1432, miR1871, miR1873, miR1875, and miR9664 (Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sheng et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, nine miRNAs have been characterized as positive regulators, including, miR159a, miR160a, miR162, miR166h-66k, miR171b, miR172a, miR398b, miR812w, and miR7695(Salvador-Guirao et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; S\u0026aacute;nchez-Mart\u0026iacute;n and Keller, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Feng et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, it is unclear whether and which miRNAs are specifically involved in PTI, ETS, and ETI in rice \u0026ndash; \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eoryzae\u003c/em\u003e interactions.\u003c/p\u003e\u003cp\u003eIn this study, we performed small RNA sequencing (small RNA-seq) under distinct experimental conditions to identify the miRNAs that may specifically act in PTI, ETS, and ETI in rice. We identified the miRNAs involved in PTI by comparing the susceptible line Lijiangxin Tuan Heigu (LTH) with or without chitin treatment, the miRNAs involved in ETS by analysing LTH infected with the virulence \u003cem\u003eM. oryzae\u003c/em\u003e strain Guy11 or the avirulent strain Δ\u003cem\u003eMoelo\u003c/em\u003e (defective in appressorium formation and invasive hyphal growth), and the miRNAs involved in ETI by examine a transgenic line expressing \u003cem\u003eR\u003c/em\u003e gene \u003cem\u003ePiz-t\u003c/em\u003e inoculated with a virulence strain RB22 or an incompatible strain carrying AvrPiz-t (RB22(AvrPiz-t)). We also examined the blast disease resistance, PTI responses, and ETI-related hypersensitive responses in the rice transgenic lines overexpressing miRNAs or by transiently expressing these miRNAs in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. Our results demonstrated that distinct miRNAs modulate PTI, ETS, and ETI during rice-\u003cem\u003eM. oryzae\u003c/em\u003e interactions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDeep-Sequencing Analysis of Small RNA Libraries\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify miRNAs involved in rice PTI, we spray-inoculated three-leaf-stage LTH seedlings with chitin or water (mock control). We collected the inoculated leaves at 0,3-, and 6-hours post-inoculation (hpi) with three biological replicates per time point for RNA extraction and constructed 15 small RNA libraries for small RNA high-throughput sequencing. The rice genome-matched miRNA reads ranged from 148,048 to 328,635 in 14 libraries except for a 3-hour chitin-treated sample with matched reads 57,178 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo identify miRNAs involved in rice ETS, we spray-inoculated three-leaf-stage LTH seedlings with either a virulent \u003cem\u003eM. oryzae\u003c/em\u003e strain Guy11 or an avirulent strain Δ\u003cem\u003eMoelo\u003c/em\u003e. Δ\u003cem\u003eMoelo\u003c/em\u003e is a mutant strain with deletion of the \u003cem\u003eMoelo\u003c/em\u003e gene, which is defective in both penetration peg formation and invasive hyphal growth(He et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We hypothesized that \u003cem\u003eΔMoelo\u003c/em\u003e fails to secret effectors into rice cells, thus significantly attenuating ETS. Therefore, the miRNAs showing differential responses to Guy11 and Δ\u003cem\u003eMoelo\u003c/em\u003e are specifically involved in ETS. LTH displayed visible disease lesions after Guy11 infection but showed invisible symptoms upon \u003cem\u003eΔMoelo\u003c/em\u003e inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The infected leaves were collected at 0, 12, and 24 hpi with three biological replicates per time points for RNA extraction. We constructed 15 small RNA libraries for high-throughput sequencing, which yielded rice genome-matched miRNA reads ranging from 47,851 to 271,369 across all libraries (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo identify miRNAs involved in rice ETI, we spray-inoculated the three-leaf-stage seedlings of a transgenic line expressing the \u003cem\u003eR\u003c/em\u003e gene \u003cem\u003ePiz-t\u003c/em\u003e in Nipponbare background (NPB (\u003cem\u003ePiz-t:HA\u003c/em\u003e) with either a compatible \u003cem\u003eM. oryzae\u003c/em\u003e strain RB22 or an incompatible RB22 strain carrying AvrPiz-t, which would trigger ETI after recognition by its cognate \u003cem\u003eR\u003c/em\u003e gene \u003cem\u003ePiz-t\u003c/em\u003e (Li et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We speculated that miRNAs involved in ETI should be differentially responsive to the two strains. As expected, \u003cem\u003ePiz-t:HA\u003c/em\u003e seedlings showed disease symptoms upon RB22 infection, whereas displayed no symptom upon RB22 (AvrPiz-t) inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The infected leaves were collected at 0, 18, and 42 hpi with three biological replicates for RNA extraction. We constructed 15 small RNA libraries for high-throughput sequencing. The rice genome-matched miRNA reads ranged from 121,727 to 285,367 in 15 libraries (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo evaluate the consistency of experimental replicates across different treatments, we performed principal component analysis (PCA) to cluster the samples in different treatment groups. The PCA results showed that the samples from the same treatment group exhibited comparable patterns and were clustered together. All PTI-related miRNAs were placed in Group 1, ETS-related in Group 2, and ETI-related in Group 3 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), indicating a high consistency among the biological replicates.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further assessed sample similarity by analysing sample-to-sample distances based on miRNA profiles. The distance heatmap revealed distinct miRNA accumulation patterns among treatment (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The size distribution analysis showed that the majority of small RNAs in our libraries were 19\u0026ndash;24 nt (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), consistent with the characteristic sizes of miRNAs and siRNAs {Mi, 2008 #343}. We identified 441 known miRNAs in these libraries (Table S2), covering most of the known rice miRNAs and validating the reliability of our sequencing data.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of miRNAs Acting in Rice PTI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe miRNAs involved in PTI were expected to be responsive to chitin in a universally susceptible rice accession LTH. We compared the miRNA reads in chitin-treated and mock samples of LTH using screening criteria including a \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fold change ˃ 2 or \u0026lt;\u0026thinsp;0.5, and the total reads in the six libraries\u0026thinsp;\u0026gt;\u0026thinsp;5. In comparison with the mock samples, no miRNAs were detected at 3-hour post-treatment (hpt), while 13 miRNAs were identified at 6 hpt, including 10 upregulated and three down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table S3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate our sequencing results, we randomly selected six miRNAs and examined their expression pattern by stem-loop real-time PCR analysis in LTH. The expression patterns of these miRNAs were basically consistent with the sequencing results: chitin induced miR1320-5p, miR398b, miR2887, and miR5814, whereas suppressed miR166g-5p and miR396e-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, Gene Ontology (GO) analysis of predicted miRNA target genes showed significant enrichment in biological process of \u0026ldquo;Response to the biotic stimulus\u0026rdquo; (Fig. S2A), implying that these miRNAs play potential roles in PTI by regulating their target genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong these miRNAs, miR1320 and miR398b have been previously characterized as positive regulators of rice blast disease resistance (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, the members of miR396 family, such as miR396a, miR396b, miR396d, and miR396h, have been identified as negative regulator (Chandran et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We then examined PAMP-triggered defence responses, including the reactive oxygen species (ROS) burst and the induction of defence-related genes, in transgenic lines overexpressing miR1320 (\u003cem\u003eOX1320\u003c/em\u003e). Compared to the Kasalath control, the \u003cem\u003eOX1320\u003c/em\u003e lines displayed enhanced ROS accumulation and increased defence-related gene-expression levels following flg22 and chitin treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G), suggesting that miR1320 enhances PTI in rice. Since miR396e-5p target the same genes as the other miR396 family members mentioned above (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.zhaolab.org/psRNATarget/\u003c/span\u003e\u003cspan address=\"https://www.zhaolab.org/psRNATarget/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we analysed PTI-related ROS burst in transgenic lines overexpressing miR396 family members (\u003cem\u003eOX396a\u003c/em\u003e, \u003cem\u003eOX396c\u003c/em\u003e, \u003cem\u003eOX396a\u003c/em\u003e, and \u003cem\u003eOX396c\u003c/em\u003e). However, the \u003cem\u003eOX396\u003c/em\u003e lines displayed a similar or slightly elevated ROS accumulation compared to the TP309 control (Fig. S3A-B), indicating that the tested miR396 members may not play a significant role in PTI and different members have different functionally preferentiality as reported in our previous study (Chandran et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of miRNAs Acting in Rice ETS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe miRNAs involved in ETS should be deferentially responsive to Guy11 and \u003cem\u003eΔMoelo\u003c/em\u003e in LTH that has PTI. To identify ETS-related miRNAs, we compared miRNA reads between Guy11-infected and \u003cem\u003eΔMoelo-\u003c/em\u003einfected LTH samples using the following screening criteria including a \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fold change ˃ 2 or \u0026lt;\u0026thinsp;0.5, and the total reads\u0026thinsp;\u0026gt;\u0026thinsp;5 across all libraries. Compared to \u003cem\u003eΔMoelo\u003c/em\u003e-infected samples that have PTI to prevent infection, 23 miRNAs were upregulated and 7 miRNAs were down-regulated in Guy11-infected samples at 12 and 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Table S4). To validate these finding, we randomly selected 10 miRNAs and examined their expression patterns. Compared to \u003cem\u003eΔMoelo\u003c/em\u003e, Guy11 infection promoted the accumulation of miR398b, miR171b, miR164d, and miR396e at both 12- and 24-hours post-inoculation (hpi), miR397b and miR408-5p at 24 hpi, whereas suppressed the accumulation of miR399d at 12 hpi, miR1862e, miR2877, and miR5814 at 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These expression patterns were consistent with the sequencing data, confirming their reliability. Moreover, GO analysis revealed that the target genes of these ETS-related miRNAs were also clustered into biological process of \u0026ldquo;Response to the biotic stimulus\u0026rdquo; (Fig. S2B), implying that these miRNAs were involved in ETS via their target genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong these upregulated miRNAs, members of the miR164 family have been characterized as negative regulators of rice blast disease resistance (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, members of the miR396 family have also been identified as negative regulators (Chandran et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These finding suggest that both miRNA families may enhance ETS, thereby compromising rice blast disease resistance. In contrast, miR171b and miR172a, which have been characterized as positive regulators, were both upregulated by Guy11 (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To determine whether these miRNAs regulate ETS, we assessed the resistance of the transgenic lines overexpressing either these miRNAs or their family members when challenged with the virulent isolate Guy11 and avirulent strain \u003cem\u003eΔMoelo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eUpon infection with the virulent strains, both \u003cem\u003eOX171b\u003c/em\u003e and \u003cem\u003eOX172a\u003c/em\u003e transgenic lines exhibited enhanced disease resistance against virulent Guy11, with fewer disease lesions and less fungal growth compared to the Nipponbare control after punch-inoculation; in contrast, these lines supported the infection of \u003cem\u003eΔMoelo\u003c/em\u003e mutant strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). Conversely, \u003cem\u003eOX396\u003c/em\u003e lines exhibited enhanced susceptibility to Guy11, with larger disease lesions and more fungal growth than the Nipponbare control, while maintaining similar symptom and fungal growth levels upon \u003cem\u003eΔMoelo\u003c/em\u003e inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). Although OX164a exhibited increased sensitivity to both Guy11 and \u003cem\u003eΔMoelo\u003c/em\u003e, the fold change in fungal biomass for \u003cem\u003eΔMoelo\u003c/em\u003e was significantly lower than that for Guy11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J). Collectively, these findings indicate that miR171b and miR172a enhances rice blast disease resistance by suppressing ETS, whereas miR396 and miR164a improve susceptibility by enhancing ETS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of miRNAs Acting in Rice ETI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe miRNAs involved in ETI should be deferentially responsive to RB22 and RB22 (AvrPiz-t) in Piz-t-expressed plants. We compared miRNA expression profiles between RB22-infected and RB22 (AvrPiz-t)-infected \u003cem\u003ePiz-t:HA\u003c/em\u003e samples, identifying differentially expressed miRNAs using screening criteria including a \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fold change ˃ 2 or \u0026lt;\u0026thinsp;0.5, and total reads\u0026thinsp;\u0026gt;\u0026thinsp;5 across all libraries. Compared to RB22-infected samples, 7 miRNAs were upregulated and 7 were downregulated in RB22 (AvrPiz-t)-infected samples at 18 or 42 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table S5). To validate these results, we randomly selected 7 miRNAs and examined their expression patterns. Compared to the virulent isolate RB22, the incompatible strain RB22 (AvrPiz-t) upregulated the expression of miR166j-3p, miR398b, and miR1882e-3p at 18 and 42 hpi, and miR1862e at 18 hpi, whereas suppressed miR396a-5p, miR397b, and miR408-5p at 18 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results are closely matched with the sequencing data, confirming their reliability. GO analysis showed that the target genes of these ETI-related miRNAs were enriched in the biological process \u0026ldquo;Secondary metabolic process\u0026rdquo; (Fig. S2C), a category tightly associated with defence responses, implying that these miRNAs may participate in ETI by regulating their target genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong these upregulated miRNAs, miR166j is from a miRNA family with members being characterized as a positive regulator of rice blast disease resistance (Salvador-Guirao et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), suggesting its positive regulatory role in ETI. In contrast, miR169 family members were known as negative regulators of blast disease resistance (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). miR169e shows high sequence similarity to miR169a, differing by two nucleotides, one at the 5\u0026rsquo; end and one at the 3\u0026rsquo; ends, targeting the same genes. Therefore, we used the transgenic lines overexpressing miR169a (\u003cem\u003eOX169a\u003c/em\u003e) for subsequent experiments. To investigate whether these miRNAs participate in ETI, we examined their effects on ETI-related cell-death by transiently co-expressing them in \u003cem\u003eN. benthamiana\u003c/em\u003e along with the rice \u003cem\u003eR\u003c/em\u003e genes \u003cem\u003ePik1\u003c/em\u003e and \u003cem\u003ePik2\u003c/em\u003e and their cognate effector \u003cem\u003eAvr-PikD\u003c/em\u003e. \u003cem\u003eAvr-PikC\u003c/em\u003e, which is not recognized by \u003cem\u003ePik\u003c/em\u003e, served as a negative control (De la Concepcion et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). miR166j accelerated \u003cem\u003eAvr-PikD\u003c/em\u003e-triggered cell death and enhanced ion leakage, whereas miR396c, and miR169a delayed cell death and reduced ion leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003eNext, we examined the roles of these miRNAs in ETI induced by incompatible \u003cem\u003eM. oryzae\u003c/em\u003e isolates. FJ81278 is an incompatible isolate to Nipponbare derived from Fujian province, the south of China (Yang, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that this isolate is also incompatible to TP309 and Kasalath (Fig. S4). Transgenic lines overexpressing miR166 were sterile, thus resistance could not be evaluated. Both \u003cem\u003eOX169a\u003c/em\u003e and \u003cem\u003eOX396\u003c/em\u003e lines exhibited compatible interactions characterized by larger lesions and more fungal biomass compared to their controls upon FJ81278 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). Consistently, these transgenic lines also displayed enhanced susceptibility to compatible strain GZ8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). These results indicated that miR169 and miR396 enhance rice susceptibility to \u003cem\u003eM. oryzae\u003c/em\u003e by suppressing ETI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBoth miR397b and miR408b-5p Enhance ETS While Suppress ETI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next identified miRNAs involved in two or three layers of rice \u0026ndash; \u003cem\u003eM. oryzae\u003c/em\u003e interactions. Venn analysis revealed that four miRNAs, including miR2877, miR396e-5p, miR398b, and miR5814, common to both PTI and ETS (Fig. S3C). \u003cem\u003eOX396\u003c/em\u003e lines displayed comparable or increased ROS accumulation relative to the TP309 control (Fig. S3A-B), suggesting that miR396e likely functions in ETS suppressing PTI.\u003c/p\u003e\u003cp\u003eFive miRNAs, namely miR397b, miR398b, miR408-5p, miR1862e, and miR1882e-3p, were common to both ETS and ETI (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We generated transgenic lines overexpressing miR397b (\u003cem\u003eOX397b\u003c/em\u003e) and miR408-5p (\u003cem\u003eOX408\u003c/em\u003e) in Nipponbare background, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). The three-leaf-stage seedlings were inoculated with \u003cem\u003eM. oryzae\u003c/em\u003e virulent strain GZ8, avirulent strain \u003cem\u003eΔMoelo\u003c/em\u003e, and incompatible isolates FJ81278. Both \u003cem\u003eOX397b\u003c/em\u003e and \u003cem\u003eOX40\u003c/em\u003e8 lines showed enhanced susceptibility to GZ8 and FJ81278 with bigger disease lesions and more fungal biomass than Nipponbare control, whereas exhibited similar incompatible lesions indifferent from the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). We further investigated their roles in effector-induced HR. When transiently co-expressed with \u003cem\u003ePiks\u003c/em\u003e and their cognate effector \u003cem\u003eAvr-PikD\u003c/em\u003e, both miRNAs greatly delayed cell death and significantly suppressed HR-triggered ion leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-H). collectively, these results indicate that miR397b and miR408-5p likely enhances rice susceptibility by promoting ETS and suppressing ETI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003emiR398b Participates in PTI, ETS, and ETI\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVenn analysis revealed that miR398b was common to all PTI, ETS, and ETS (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We previously have characterized it as a positive regulator of blast resistance and PTI {Li, 2019 #162}. However, its roles in ETS and ETI remained unexplored.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then analysed the roles of miR398b in PAMP-induced ROS burst and ETI-related HR, as well as the resistance to virulent and incompatible strains. The transgenic lines overexpressing miR398b (\u003cem\u003eOX398b\u003c/em\u003e) showed higher and more ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eOX398b\u003c/em\u003e displayed enhanced resistance to the virulent strain NC-10 and GZ8, as well as the incompatible isolate FJ81278 with smaller disease lesions and less fungal biomass than the Kasalath control (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D), while similar response to the avirulent strain \u003cem\u003eΔMoelo\u003c/em\u003e. Consistently, miR398b greatly delayed cell death and significantly suppressed HR-triggered ion leakage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F). Taken together, these results indicated that miR398b enhanced rice blast disease resistance by improving both PTI and ETI, presumably via suppressing ETS.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we screened out 48 miRNAs specifically in PTI, ETS, and/or ETI (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Among these miRNAs, 12 miRNAs, including miR164, miR166, miR168, miR169, miR171, miR172, miR396, miR397b, miR398b, miR444, miR1320-5p, and miR1432-5p, have been previously characterized as regulators of rice blast disease resistance. We functionally characterized nine of these known plus one previously uncharacterized miRNA, miR408-5p, across all the three layers of rice \u0026ndash; \u003cem\u003eM. oryzae\u003c/em\u003e interaction. Our results indicate that these miRNAs orchestrate rice blast resistance through distinct mechanisms. miR1320 enhances resistance via regulating PTI; miR171b and miR172a enhances, whereas miR164 and miR396 families suppresses resistance via regulating ETS; miR166 enhances, whereas miR169 and miR396 suppress resistance via regulating ETI. Moreover, miR397b and miR408-5p suppress resistance via enhancing ETS while suppressing ETI; miR398b enhances resistance via improving PTI and ETI, while suppressing ETS. Our results reveal a complicated regulatory network that miRNAs collaboratively modulate multi-layered defence responses, thereby regulating blast disease resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the miRNAs identified as PTI regulators through deep sequencing, miR1432 have been characterized as a negative regulator of PTI in rice. Compared to the Nipponbare control, the transgenic lines overexpressing miR1432 showed compromised ROS burst and callose deposition induced by PAMPs, whereas the transgenic lines overexpressing its target gene \u003cem\u003eEF-hand family protein 1\u003c/em\u003e (\u003cem\u003eEFH1\u003c/em\u003e) exhibited enhanced defence responses (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, deep sequencing data showed that chitin treatment enhances the accumulation of miR1432 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), confirming that miR1432 facilitated rice susceptibility via suppressing PTI. However, the mechanism by which miR1432-EFH1 regulatory module acts in PTI needs further investigation.\u003c/p\u003e\u003cp\u003eIn this study, we identified several miRNAs by deep sequencing, whose family members were previously characterized. Notably, miR396e-5p was identified as a regulator of both PTI and ETS. In a previous study, we have characterized the roles of another four miR396 family members in rice blast disease resistance, including miR396a-5p, miR396c-5p, miR396d-5p, and miR396h-5p(Chandran et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although these family members and miR396e-5p exhibited 1\u0026ndash;2 nucleotide variations, they maintain functional conservation through target identical genes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.zhaolab.org/psRNATarget/\u003c/span\u003e\u003cspan address=\"https://www.zhaolab.org/psRNATarget/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eOX396\u003c/em\u003e lines showed comparable ROS burst similar to the Nipponbare control (Fig. S3A-B), indicating that miR396 likely does not modulate PTI. Conversely, all the overexpressing lines of the four family members exhibited enhanced ETS phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H), indicating that miR396 members likely facilitate rice susceptibility by improving ETS. In rice, miR396 targets 12 growth-regulating factors (GRFs) that orchestrate diverse physiological processes, such as grain size determination, inflorescence development, plant architecture determination, blast disease resistance, salt tolerance, and brown planthopper resistance (Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chandran et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dai et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, it is worth further investigation of the downstream signalling pathways of miR396-GRFs module in ETS.\u003c/p\u003e\u003cp\u003eAnother identified miRNA, miR169e, differs from miR169a by two-nucleotides variation, while maintain functional conservation through target identical genes nuclear transcription factor Y-A family members (\u003cem\u003eNF-YAs\u003c/em\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.zhaolab.org/psRNATarget/\u003c/span\u003e\u003cspan address=\"https://www.zhaolab.org/psRNATarget/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Therefore, we hypothesized that miR169e and miR169a share functional conservation in regulating rice blast resistance. We previously demonstrated that miR169a negatively regulates blast disease resistance (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), although its molecular mechanism remained unclear. In this study, we found that miR169a overexpressing lines exhibited enhanced susceptibility to both compatible and incompatible strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F), suggesting that miR169 family members facilitate rice susceptibility by suppressing ETI. Notably, NF-YAs suppress jasmonic acid (JA)-mediated antiviral defence and promote susceptibility to rice stripe virus (RSV) and Southern rice black-streaked dwarf virus (SRBSDV) (Tan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Whether miR169-\u003cem\u003eNF-YAs\u003c/em\u003e module regulates ETI in rice via JA signalling is unknown and need further study.\u003c/p\u003e\u003cp\u003eIn this study, we identified and characterized miR408-5p as novel regulator of both ETS and ETI. miR408 is a highly conserved miRNA family in plant widely modulating stress response, growth, and developments. In wheat, Tae-miR408 suppresses wheat responses to high-salinity, heavy cupric stress, and stripe rust by targeting a gene encoding a chemocyanin-like protein (Feng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In maize, miR408b negatively regulates resistance against \u003cem\u003eFusarium verticilliodes\u003c/em\u003e, the agent of maize Fusarium ear rot (FER) (Zhou et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Here we showed that \u003cem\u003eOX408\u003c/em\u003e lines exhibited susceptibility to the incompatible strain and enhanced susceptibility to virulent strain compared to the Nipponbare control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E), suggesting that miR408 likely enhances rice susceptibility by suppressing ETI. However, \u003cem\u003eOX408\u003c/em\u003e lines showed no significant difference in symptom development when challenged with Δ\u003cem\u003eMoelo\u003c/em\u003e, which impaired in appressorium formation and invasive hypha development (both required for effector secretion), indicating that miR408 likely does not participate in PTI.\u003c/p\u003e\u003cp\u003eAmong the 48 miRNAs identified in this study, the function of 36 miRNAs in rice blast disease resistance remain uncharacterized. Notably, miR1862e emerged as a particularly promising candidate based on high accumulation in rice and identification in both ETS and ETI, suggesting its potential importance in rice-\u003cem\u003eM. oryzae\u003c/em\u003e interactions. However, the roles of miR1862e were not ever been reported. Given this finding, future research should prioritize functional characterization of miR1862e in disease resistance and related molecular mechanism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we identified miRNAs involved in PTI, ETS, and ETI during the rice\u0026ndash;\u003cem\u003eM. oryzae\u003c/em\u003e interaction and selected a total of 48 miRNAs for further analysis. We functionally characterized the roles of 10 of these miRNAs across all three layers of immunity. Our results reveal a complex regulatory network in which miRNAs collaboratively modulate multi-layered defence responses, thereby regulating blast disease resistance. These findings provide theoretical support and genetic resources for disease-resistant rice breeding.\u003c/p\u003e"},{"header":"Experimental Procedures","content":"\u003cp\u003e\u003cb\u003eGrowth Condition and Plant Material\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rice (\u003cem\u003eOryzae sativa\u003c/em\u003e) plants were used in this study, including \u003cem\u003eJaponica\u003c/em\u003e accessions Lijiangxin Tuan Heigu (LTH), Nipponbare, \u003cem\u003ePiz-t\u003c/em\u003e lines that expressing HA-fused \u003cem\u003eR\u003c/em\u003e gene \u003cem\u003ePiz-t\u003c/em\u003e in Nipponbare background (\u003cem\u003ePiz-t:HA\u003c/em\u003e), Indica accessions TP309, Kasalath, and the transgenic lines overexpressing miRNAs in indicated background. These rice plants were grown in a plant growth chamber under the controlled conditions with a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;at 2\u0026deg;C Day/night, 70% relative humidity and a 14/10-h light/dark period.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathogen Infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eM. oryzae\u003c/em\u003e strains or isolates were used for blast infection, including Guy11, GZ8, NC-10, FJ81278, Δ\u003cem\u003eMoelo\u003c/em\u003e, RB22, and RB22 expressing an \u003cem\u003eM. oryzae\u003c/em\u003e effector (\u003cem\u003eAvrPiz-t\u003c/em\u003e). These strains were grown on oat-tomato agar medium for 2 weeks at 28\u0026deg;C with a 12/12-h day/night treatment for sporulation. After getting rid of the surface mycelia with distilled water, the plates were further incubated for 3 days with consistent light treatment to promote sporulation. Spores were collected with concentration 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e conidia mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The five-leaf-stage seedlings were spray inoculated for small RNA library construction or punch-inoculated for resistance assay. For punch inoculation, the leaves were slightly wounded with a mouse-ear punch, and the conidia suspension was punch-inoculated on wounded sites. The disease symptoms were observed at 5\u0026ndash;7 days post-inoculation (dpi). The relative fungal biomass on infected leaves was measured according to a previous report (Park et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The infected leaves were collected for DNA extraction. The relative fungal biomass was determined using the DNA amount of fungal \u003cem\u003eMoPot2\u003c/em\u003e against rice DNA amount of ubiquitin (UBQ) through q-PCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLibrary Construction, Sequencing, and Bioinformatics Analysis of Small RNAs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe small RNA library construction and Illumina sequencing were conducted following a previous report with some revision(Mi et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In a summary, the total RNA extracted from the different treatment samples were used for small RNA library construction and RNA-sequencing (Oebiotech Company). Each treatment contains three repeats. The small RNA reads with length over 16 were mapped to the rice genomes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; version 7.0). The perfect genome-matched small RNAs were analysed and the normalized abundance of small RNAs was calculated as reads per million.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasmid Construction and Genetic Transformation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo construct the transgenic lines overexpressing miR397b and miR408-5p, the sequence on the \u003cem\u003eMIR397b\u003c/em\u003e gene containing 278 bp upstream to 281 bp downstream and \u003cem\u003eMIR804-5p\u003c/em\u003e gene containing 275 bp upstream to 276 bp downstream sequence was amplified from NPB genomic DNA with primers miR397b-F, miR397b-R and miR408-5p-F, and miR408-5p-R (Supplementary Table S10). PCR products were cloned into the binary vector 35S-pCAMBIA1300. The constructs were transformed into Nipponbare via \u003cem\u003eAgrobacterium tumefaciens-\u003c/em\u003emediated transformation. Transgenic plants were screened by PCR with the forward primer 35S-F and the specific reverse primers miR397b-R and miR408-5p-R (Supplemental Table S10). The positive transgenic lines were used for resistance analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA Extraction and Gene Expression Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor defence-related gene expression assay, three-leaf-stage rice seedlings of indicated lines were spray-inoculated with chitin or flg22, and the samples were collected at indicated time points. For miRNA transgenic line identification, the leaves from the T0 transgenic lines were collected. Total RNAs were extracted from the collected leaves using TRIzol reagent (No. 7E2582B5, Vazyme, NanJing, China). To determine the expression of PTI-related genes, the first-strand cDNA was synthesized from 1 \u0026micro;g of total RNA using PrimeScript RT Reagent Kit with gDNA Eraser following the manufacturer\u0026rsquo;s instructions (RR047A, TaKaRa). RT-qPCR was performed using specific primers and SYBR Green mix (QuantiNova SYBR Green PCR Kit; Qiagen) with the BIO-RAD C1000TM Thermal Cycler (Bio-Rad Inc., China). The rice ubiquitin (\u003cem\u003eUBQ\u003c/em\u003e) gene was used as an internal reference to normalize the relative expression levels of genes. To examine the amount of miR408 and miR397b, total RNA was reverse-transcribed using a miRNA-specific stem-loop RT primer (Table S10), and the RT product was subsequently used as a template for RT-qPCR by using miRNA-specific forward primers and the universal reverse primer (Table S10). snRNA U6 was used as an internal reference to normalize the relative amounts of miRNAs. RT-qPCR analyses were performed with three technical replicates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReactive Oxygen Species (ROS)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe leaves of five-leaf-stage rice seedling were selected for ROS burst assay. The leaves were cut into 5mm-width squares with 1-mm-width stripes and incubated in 200 \u0026micro;L water in a 96-well plate for 12\u0026ndash;16 h. Then, the squares were treated with or without 20 \u0026micro;g/mL chitin or 1 \u0026micro;M flg22 in 200 \u0026micro;L buffer containing 10 \u0026micro;g/mL horseradish peroxidase (Sigma-Aldrich, Shanghai, China) and 20 mM L-012 (Wako, Japan). The production of ROS was detected using a GLOMAX96 Microplate Luminometer (Promega Biotech Co., Ltd, Beijing, China) for 30\u0026ndash;60 min and determined as relative luminescence units.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHypersensitive Response (HR) and Conductivity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the cell death regulated by miRNAs, the cDNA sequences of \u003cem\u003ePik1\u003c/em\u003e and \u003cem\u003ePik2\u003c/em\u003e were amplified from ZH11 cDNA using indicated primers (Supplementary Table S10). Avr-PikC and Avr-PikD cDNA sequences were amplified from Guy11 cDNA using indicated primers (Supplementary Table S10). The amplified PCR products were cloned into the \u003cem\u003eKpn\u003c/em\u003e1 and \u003cem\u003eSac\u003c/em\u003e1 restriction sites of the pCAMBIA1300 vector and transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens GV3101. These strains were grown in lysogeny broth (LB) liquid media at 28 \u0026ordm;C overnight and resuspended in buffer (10 mM 2-(N-morpholino) ethanesulfonic acid [MES] [pH 5.5\u0026ndash;5.7], 10 mM MgCl2, 200 mM acetosyringone) and injected into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves for transient expression. The phenotype was observed and the photo were capture with UV-light after 4\u0026ndash;5 dpi. Then, the infected leaves were cut with a holler cutter and the leaf dices were transferred into the 10 ml tubes with sterilized ddH\u003csub\u003e2\u003c/sub\u003eO and kept at room temperature overnight. Then the tubes were boiled at 100\u0026deg;C for 5 minutes and kept for cooling at room temperature for 2 hours. After cooling, the conductance was measured with the help of a conductivity meter.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePAMP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;pathogen-associated molecular pattern\u003c/p\u003e\n\u003cp\u003ePTI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;PAMP-triggered immunity\u003c/p\u003e\n\u003cp\u003eETS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; effector-triggered susceptibility\u003c/p\u003e\n\u003cp\u003eETI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;effector-triggered immunity\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eHR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hypersensitive response\u003c/p\u003e\n\u003cp\u003eRT-qPCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Real-time quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eLTH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lijiang xin Tuan Heigu\u003c/p\u003e\n\u003cp\u003eNPB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Nipponbare\u003c/p\u003e\n\u003cp\u003eTP309 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Taipei309\u003c/p\u003e\n\u003cp\u003eGRFs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Growth-regulating factors\u003c/p\u003e\n\u003cp\u003eNF-YAs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Nuclear transcription factor Y-A family members\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and material mentioned in this study is available upon requirement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 32121003, 32172417, 32372553,\u0026nbsp;and 32401845), Science and technology department of Sichuan province (2025ZNSFSC1115, SCCXTD-2024-SD-4, and 2021YFYZ0021-4-2), and the Open Research Fund of State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China (SKL-ZD202202, SKL-ZY202202, SKL-ZY202205, and SKL-ZY202209).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW-M. Wang and Y. Li designed the experiments. S-H. Bhutto, Y. Z., H. S., X. H., X-Y. X., and Y. Yang conducted the experiments. Y-H. Z., H-S D., S-Y. Z., and D-Q. Li conducted the field trials. X-M. Y., H. W., G-B L., Z-X. Z., and J-W. Zhang analysed the data. W-M. W. and Y. Li wrote the paper. Y-Y. H. and M-I. Khaskheli discussed the results and commented on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Prof. Guo-Liang Wang at Ohio State University for providing RB22 and RB22(AvrPiz-t) strains, as well as the \u003cem\u003ePiz-t:HA\u003c/em\u003e transgenic lines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaldrich P, Campo S, Wu MT, Liu TT, Hsing YL, San Segundo B (2015) MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors. 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Genomics Proteom Bioinf 18:241\u0026ndash;255\u003c/span\u003e\u003c/li\u003e\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":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"rice blast disease, miRNAs, Small RNA-sequencing, PTI, ETS, ETI","lastPublishedDoi":"10.21203/rs.3.rs-7070474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7070474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRice blast disease is one of the most destructive rice diseases worldwide. Rice MicroRNAs (miRNAs) play an essential role in immunity against blast fungus \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e. However, it remains unclear which miRNAs are involved in the three layers of rice-\u003cem\u003eM. oryzae\u003c/em\u003e interaction, including pathogen associated molecular patterns (PAMPs)-triggered immunity (PTI), effector-triggered susceptibility (ETS), and effector-triggered immunity (ETI). In this study, we performed small RNA-sequencing to systemically identify miRNAs regulating PTI, ETS, and ETI in rice-\u003cem\u003eM. oryzae\u003c/em\u003e interaction. A totally 441 miRNAs were identified, with 13, 30, and 14 miRNAs screened out and classified as regulators of PTI, ETS, and ETI, respectively. We investigated and confirmed the roles of 9 previously reported miRNAs and an uncharacterized miRNA, miR408-5p, in the three interaction processes. We demonstrated that miR1320-5p positively regulated PTI; miR396 family members and miR164a improved, whereas miR171b and miR172a suppressed ETS; miR166a enhanced, whereas miR169a and miR396 family members suppressed ETI. Moreover, we demonstrated that miR397b and miR408-5p enhanced rice susceptibility by promoting ETS and suppressing ETI; miR398b enhanced rice resistance by promoting both PTI and ETI while suppressing ETS. Our findings figured a miRNA-mediated regulatory network in which distinct miRNAs modulate PTI, ETS, and ETI against \u003cem\u003eM. oryzae\u003c/em\u003e. This study provides theoretical support and genetic resources for disease-resistant breeding in rice.\u003c/p\u003e","manuscriptTitle":"Identification of MicroRNAs Involved in Different Layers of rice-Magnaporthe oryzae interaction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 18:00:32","doi":"10.21203/rs.3.rs-7070474/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-20T14:26:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T01:44:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T12:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98576696685594964932588202878982734635","date":"2025-08-06T10:46:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159520240146514362156868981357987566996","date":"2025-08-06T03:35:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311249665033210766437457230625236833214","date":"2025-08-06T00:24:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T15:41:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T04:04:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-09T04:03:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2025-07-08T04:34:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef321c87-60ad-4cbb-a287-bfdd0bff4141","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:34:33+00:00","versionOfRecord":{"articleIdentity":"rs-7070474","link":"https://doi.org/10.1186/s12284-025-00855-8","journal":{"identity":"rice","isVorOnly":false,"title":"Rice"},"publishedOn":"2025-10-24 16:16:47","publishedOnDateReadable":"October 24th, 2025"},"versionCreatedAt":"2025-07-17 18:00:32","video":"","vorDoi":"10.1186/s12284-025-00855-8","vorDoiUrl":"https://doi.org/10.1186/s12284-025-00855-8","workflowStages":[]},"version":"v1","identity":"rs-7070474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7070474","identity":"rs-7070474","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}