Rice (Oryza sativa L.) was domesticated approximately 10,000 years ago in the lower Yangtze Valley in China. From there, it spreads across Asia, Africa, Europe, and the Americas, and now serves as a staple crop for more than half of the world’s population (Cheng et al., 2013; Gutaker et al., 2020). However, rice production suffers from numerous pests and pathogens. Among them, the brown planthopper (BPH; Nilaparvata lugens Stål) is considered extremely destructive (Bottrell and Schoenly, 2012; Cheng et al., 2013; Jing et al., 2017). BPH feeds on phloem sap and causes dwarfing, wilting, browning, drying, and ultimately death in severe cases (Wang et al., 2008). In addition, BPH serves as a vector for viral diseases, resulting in significant yield shortfalls and economic losses (Cheng et al., 2013).

Over the course of an evolutionary arms race between these two species, rice has developed sophisticated defensive mechanisms against BPH (Cheng et al., 2013), including both basic defenses and resistance (R) gene-mediated defenses. To date, approximately 40 major BPH resistance genes have been identified in cultivated and wild rice species (Wang et al., 2023). Molecular cloning and functional characterization of BPH resistance genes have clarified the molecular mechanisms of rice resistance to BPH (Zheng et al., 2021). One mechanism involves the occlusion of sieve tubes with callose, which is a common plant defense against sap-sucking insects. This mechanism is the most effective in rice varieties carrying BPH resistance genes such as B5 (carrying Bph14 and Bph15), RI35 (carrying Bph14), and YHY15 (carrying Bph15) (Hao et al., 2008). In susceptible varieties such as Taichung Native1 (TN1), β-1,3-glucanases (which are only weakly induced in resistant plants) decompose the deposited callose and thereby facilitate continuous feeding by BPH (Hao et al., 2008). Bph14 encodes a typical CC-NB-LRR protein which interacts with the transcription factors (TFs) OsWRKY46 and OsWRKY72 to increase the expression of the receptor-like cytoplasmic kinase gene RLCK281 and the callose synthase gene by binding to their promoters (Du et al., 2009; Hu et al., 2017). Bph15 is located on the short arm of chromosome 4 and is composed of a gene cluster encoding three plant lectin receptor-like kinase proteins (LecRLKs) (Yang et al., 2004; Lv et al., 2014; Xiao et al., 2016). However, the precise mechanism by which Bph15 confers resistance to BPH is unknown.

Phytohormones are thought to play pivotal roles in the interaction of rice plants and BPH herbivory, including salicylic acid (SA), ethylene (ET), jasmonic acid (JA), cytokinin (CK), brassinosteroid (BR), and abscisic acid (ABA). The SA pathway contributes to the immune response against piercing–sucking insects and is involved in Bph6-, Bph9-, Bph14-, and Bph29-mediated resistance in rice (Du et al., 2009; Wang et al., 2015; Zhao et al., 2016; Hu et al., 2017; Guo et al., 2018). Antagonistically, ET negatively regulates BPH resistance in rice (Lu et al., 2011, 2014; Ma et al., 2020). However, the role of JA in rice resistance to BPH remains controversial as the silencing of different genes related to JA biosynthesis and signaling results in diverse impacts on BPH resistance. For example, silencing 9-lipoxygenase (OsLOX9/OsHI-LOX, a JA biosynthesis-related gene) enhances BPH resistance, suggesting that JA negatively regulates BPH resistance (Zhou et al., 2009). On the other hand, silencing coronatine insensitive 1 (OsCOI1, a JA receptor gene) has no effect on BPH resistance (Ye et al., 2012). According to studies in allene oxide cyclase (AOC, a JA biosynthesis-related enzyme)- and Myelocytomatosis protein 2 (MYC2, a bHLH TF in the JA pathway)-knockout mutant rice, JA appears to be a positive regulator of BPH resistance (Xu et al., 2021). The exogenous application of JA similarly suggests this (Guo et al., 2018). Recent research also suggests that CK may positively regulate BPH resistance in a JA-dependent manner (Zhang et al., 2022). Conversely, BR promotes BPH susceptibility by modulating SA and JA signaling (Pan et al., 2018). ABA enhances BPH resistance by promoting callose formation (Dinh et al., 2013; Liu et al., 2017) and synergizes with JA to stimulate the expression of TFs in BPH-infested rice (Li et al., 2022). Furthermore, a coordinated CK, SA, and JA signaling network has been found to be activated in Bph6-near isogenic lines (NILs) (Guo et al., 2018). Taken together, these findings suggest that the various phytohormones play diverse roles in the BPH defense response, and that there is complex crosstalk between them.

BPH biotypes with increased virulence have emerged in response to pressures imposed by these defense mechanisms, which are capable of overcoming resistance conferred by major resistance genes. Biotype 1 BPH, which exhibits low virulence on resistant rice varieties, is widely distributed across southeast Asia and primarily parasitizes susceptible varieties such as TN1 (Alam & Cohen, 1998). Rearing biotype 1 BPH on resistant rice variety YHY15 (carrying Bph15) for several years resulted in the development of highly-virulent biotype Y BPH, which are able to overcome resistance conferred by Bph15 (Jing et al., 2011). Similarly, after force-feeding 40 generations of local BPH using resistant IR56 rice (carrying Bph3), the resulting BPH population (IR56-BPH) was able to overcome Bph3-conferred resistance (Zheng et al., 2016). In addition, the Mudgo BPH population has been reported to cause substantial damage to Mudgo rice plants (carrying Bph1) (Ji et al., 2013; Wan et al., 2019). In response to selective pressures imposed by these resistance genes, BPH populations accumulate adaptations over generations which eventually allow them to overcome such resistance mechanisms. In effect, this process constitutes a loss of resistance in formerly-resistant rice varieties against them. However, the effectiveness of resistance strategies used by different rice varieties against BPH populations with varying levels of virulence requires further investigation. Such combinations of avirulent/virulent BPH and resistant rice provide ideal models for studying resistance adaptation mechanisms.

Just as plants have evolved intricate defense mechanisms to protect themselves against herbivorous insects, insects have in turn developed strategies to overcome plant defenses (Liu Q. et al., 2021). Interactions between plants and insects involve an array of molecular, biochemical, and physiological processes occurring at multiple levels. Multi-omics analyses integrate genomics, transcriptomics, proteomics, and metabolomics, as well as other “omics” approaches, in order to clarify the intricate signaling pathways, molecular responses, and biochemical processes involved in the dynamic interplay between plants and insects (Wang et al., 2020; Shi et al., 2023). Transcriptional profiling has aided our understanding of the defense mechanisms utilized by rice against BPH. For example, research suggests that BPH infestation results in the upregulation of genes involved in signaling, oxidative stress, pathogen-related response, and macromolecule degradation, as well as the downregulation of genes associated with flavonoid biosynthesis, photosynthesis, and cell growth (Zhang et al., 2004; Yuan et al., 2005; Wang et al., 2008). In addition, microarray analyses of BPH-infested Rathu Heenati and TN1 rice underscore the importance of TFs and phytohormones in the defense response (Wang et al., 2012; Li et al., 2017).

MicroRNAs (miRNAs) are small (approximately 21 nt in length) regulatory RNAs produced through endonucleolytic processing of hairpin precursors (Axtell and Meyers, 2018). miRNAs regulate gene expression by binding to complementary sequences in mRNA molecules, resulting in degradation and/or translational inhibition (Bartel, 2009). In plants, miRNAs are involved in various processes such as phytohormone signaling; abiotic and biotic stress response (Zhang et al., 2013, 2016; Li et al., 2017; Natarajan et al., 2018; Salvador-Guirao et al., 2018; Zhang et al., 2018; Liu X. et al., 2021); and leaf, flower, shoot, root, and vascular tissue development (Marin et al., 2010; Peng et al., 2014; Mangrauthia et al., 2017). However, only a few miRNAs have been found to play roles in the insect-plant interaction (Jing et al., 2023). For example, in an investigation of resistant and susceptible rice varieties, the BPH-responsive miRNAs miR156 and miR396 were found to negatively regulate BPH resistance by regulating JA and flavonoid biosynthesis, respectively (Wu et al., 2017; Ge et al., 2018; Dai et al., 2019). Studies of BPH-responsive mRNA and miRNA transcriptomes have uncovered certain universal responses of rice to BPH infestation (Li et al., 2017; Wu et al., 2017; Nanda et al., 2020). Additionally, integrated expression profiling of miRNAs and target genes associated with the BPH-rice interaction has been conducted (Tan et al., 2020). Such combined miRNA and mRNA analyses will be key to unraveling the transcriptional responses of rice to BPH infestation.

In this study, we employed high-throughput sequencing to analyze the mRNA and miRNA expression profiles of YHY15 rice seedlings infested with either biotype 1 BPH (avirulent population) or biotype Y BPH (virulent population). Furthermore, we combined sequence analysis and physiological assays to reveal the underlying resistance mechanisms of rice against BPH. The findings presented in this work will provide a valuable resource for further genome-wide investigations of BPH-responsive genes, as well as studies of Bph15-mediated resistance. Moreover, these findings improve our understanding of the intricate interactions between rice and BPH, and may be used in the development of effective BPH management strategies.

The management of rice plant resistance to BPH infestation will be crucial for effective pest management. Understanding the defense strategies employed by resistant rice varieties against both avirulent and virulent BPH populations can provide valuable insights for developing effective pest control strategies. Transcriptomics, which can quantify changes in gene expression and associated regulatory mechanisms, can aid in unraveling these complexities. In plants, miRNAs are involved in various developmental processes and play significant roles in abiotic and biotic stress responses (Zhang et al., 2013; Kumar, 2014; Zhang et al., 2016; Li et al., 2017; Mangrauthia et al., 2017; Natarajan et al., 2018). Specifically, miRNAs regulate targeted gene expression by binding complementary sequences in mRNA molecules, resulting in degradation and/or inhibited translation (Bartel, 2009; Marin et al., 2010; Peng et al., 2014). Integrated mRNA and miRNA transcriptomics analyses have been used to identify miRNA-mRNA networks associated with developmental processes and insect defense (Tan et al., 2020; Li et al., 2021; Mei et al., 2021). However, little attention has been paid to defense strategies employed by resistant rice against differentially virulent BPH populations. In this study, we employed an integrated mRNA and miRNA transcriptomics approach to characterize the defense responses of resistant YHY15 rice (contains Bph15) to infestation with avirulent (biotype 1) and virulent (biotype Y) BPH. Our results revealed that YHY15 rice seedlings exhibited distinct responses under the two infestation scenarios ( Figure 1 ). YHY15 rice is highly resistant to biotype 1 BPH and susceptible to biotype Y BPH, which is in accordance with previous reports (Yang et al., 2004; Jing et al., 2011; Guan et al., 2022).

The BPH-resistance gene Bph15 has been widely applied in rice breeding programs, although the molecular mechanisms underlying Bph15-mediated resistance remain unclear (Lv et al., 2014; Zheng et al., 2021). Previous RNA-seq studies of Bph15 introgression lines and recipient lines before and after BPH infestation have identified key defense mechanisms associated with phytohormone signaling, mitogen-activated protein kinase (MAPK) cascades, receptor kinases, protein post-translational modifications, TFs, Ca²⁺ signaling, and pathogenesis-related proteins (Lv et al., 2014). In addition, 20 upregulated and 3 downregulated miRNAs were identified in resistant rice variety P15 (containing Bph15) compared to susceptible rice variety PC (recipient line) (Wu et al., 2017). Combined with the mRNA transcriptome data, the 67 potential targets of these miRNAs were related to resistance responses to avirulent BPH, including abiotic and biotic stimuli, regulation of plant hormones (GA, SA, ET, and CK), cellulose biosynthesis, amino acid biosynthesis, and protein folding (Cheng et al., 2013; Lv et al., 2014; Wu et al., 2017). Here, comprehensive analysis of miRNA-seq and mRNA-seq data shed light on the underlying mechanisms responsible for the contrasting responses of YHY15 rice to BPH biotypes with different virulence.

Plant defense against BPH is a dynamic and sophisticated process which involves many levels of organizational and functional complexity (Barah and Bones, 2015; Erb and Reymond, 2019). During feeding, the BPH stylet transiently punctures the epidermis and then penetrates the plant cell wall. Subsequently, the insect salivates into the cells and ingests the phloem sap (Hao et al., 2008). According to electronic penetration graph (EPG) waveform recordings, BPH begin feeding on phloem sap within 1-3 hours of settling on rice plants (Hao et al., 2008). A greater number of miRNAs were found to be upregulated in biotype 1 BPH-infested resistant P15 rice (a Bph15 introgression line) than in susceptible PC rice (recipient line) during the early infestation stage (6 h) when the plants had not yet been severely damaged (Wu et al., 2017). In another report, the inducible BPH defense responses (indicated by upregulated DEGs and DEMs) were more robust during the early feeding stages (e.g., 6, 12, and 24 h) in resistant BPH6G rice (BPH6-transgenic rice) than in susceptible Nipponbare rice (wild type, WT) (Tan et al., 2020). Moreover, a miRNA profiling was conducted on resistant IR56 rice (carrying Bph3) under separate infestations of a virulent IR56-BPH and an avirulent TN1-BPH (Nanda et al., 2020). This study revealed that BPH feeding caused significant alterations in miRNA expression profiles of IR56 rice, with a greater number of miRNAs showing downregulation when IR56 rice was infested with TN1-BPH. However, the distinct mechanisms underlying rice plant responses to BPH of varying levels of virulence remains unclear. Here, resistant YHY15 rice plants were exposed to avirulent (biotype 1) and virulent (biotype Y) BPH. Notably, DEMs were only identified in rice plants infested with biotype Y BPH ( Figure 2A ). In addition, many more DEGs were identified in rice plants infested with biotype Y BPH, regardless of the infestation time ( Figure 2E ). Together, these results suggest that biotype Y BPH elicit more intense defense responses in YHY15 rice. DaEGs related with cytochrome P450 (KEGG) were more common at the early infestation stage (6 h) in rice infested with both types of BPH ( Figure 2F ; Supplementary Figures 1A, B ). At the later infestation stage (48 h), overlapping DEGs in R0 vs RT48 and R0 vs RY48 were enriched in photosynthesis-related pathways ( Figure 2F ; Supplementary Figures 3A, B ). Specifically, DEGs in R0 vs RY6 were mainly enriched in photosynthetic organisms and DNA replication proteins ( Figure 2F ; Supplementary Figures 2A, B ), indicating that damage to the photosynthetic system occurs earlier in biotype Y BPH-infested rice. Taken together, compared with biotype 1 BPH, infestation with biotype Y BPH results in more serious damage and induces more intense transcriptional responses in YHY15 rice.

In plants, including rice, phytohormone signaling is widely known to be involved in insect defense and resistance (Liu et al., 2016; Jing et al., 2023; Liu Q. et al., 2021). Among phytohormones, JA is critical to the regulation of plant defenses against insect herbivores (Thaler et al., 2012; Howe et al., 2018). Other phytohormones, such as SA, ET, BR, ABA, and CK are also involved in plant responses to herbivory through cross-talk with JA signaling (Bruessow et al., 2010; Schäfer et al., 2015; Pan et al., 2018; Ma et al., 2020; Li et al., 2022; Zhang et al., 2022). In this study, we found that infestation with differentially virulent BPH populations resulted in noticeable effects on the expression of genes associated with phytohormone signaling in YHY15 rice plants. Specifically, infestation with biotype 1 BPH induced JA- and ABA-related signaling pathways, but suppressed ET- and BR-related signaling ( Figure 5 ). While, infestation with biotype Y BPH induced more moderate expression of JA- and ABA-related pathways. These results suggest that although infestation with virulent biotype Y BPH results in extensive damage to YHY15 rice plants, they exhibit weaker JA- and ABA-mediated defense responses. These findings are in line with previous research suggesting that IR56 BPH can overcome Bph3-mediated resistance by suppressing the transcription of defense-responsive MAPK pathways, phytohormone biosynthesis, and secondary metabolite production (Nanda et al., 2018). Previous studies also suggest that oral secretions, digestive and detoxifying enzymes, and endosymbionts can help insect herbivores adapt to host plants (Simon et al., 2015; Yates and Michel, 2018). We therefore speculate that specific BPH effectors may interact with Bph15 and affect Bph15-mediated immunity in rice. However, further studies will be required to test this hypothesis.

The DEMs and their associated mRNAs identified in this study may potentially play roles in the response of rice to BPH infestation. Among the identified DEMs, miR156, miR5076, miR1856, miR398, miR5072, miR5079, miR408, miR2873, and miR169 exhibited significant variation in amplitude ( Supplementary Table 3 ). In addition, many of them have been implicated in various plant developmental processes and stress responses (Li et al., 2017b) (Sharma et al., 2015; Zhao et al., 2017; Lin et al., 2018; Liu et al., 2019; Liebsch and Palatnik, 2020; Li et al., 2020; Liu X. et al., 2021; Gao et al., 2022; Pachamuthu and Hari Sundar, 2022; Zhao et al., 2022). The altered expression of these miRNAs may enhance BPH resistance in YHY15 rice. Combined with mRNA transcriptome data ( Table 2 ), we found that the miR156-JA, miR396b–GRF8–F3H–flavonoid, and miR398b-callose deposition pathways may contribute to the resistance of YHY15 rice to biotype Y BPH. Physiological tests verified the increased deposition of callose in biotype Y BPH-infested rice ( Figure 7 ). Notably, these miRNA-mediated responses only occurred in rice infested with biotype Y BPH, which suggests that they may contribute to the differential resistance of rice against BPH populations with varying levels of virulence.

Finally, research suggests that numerous TFs are involved in the rice response to BPH infestation. For example, OsMYB30 (an R2R3 MYB TF) induces the expression of phenylalanine ammonia-lyase (PAL) enzymes, thereby improving BPH resistance in rice (He et al., 2020). OsMYB22 promotes rice resistance by affecting flavonoid biosynthesis (Sun et al., 2023). The bHLH protein MYC2 is involved in JA-mediated insect resistance (Schweizer et al., 2013; Xu et al., 2021). OsWRKY45, OsWRKY53, OsWRKY70, and OsWRKY89 also mediate herbivore resistance (Chujo et al., 2007; Wang et al., 2007; Hu et al., 2015; Li et al., 2015; Huangfu et al., 2016). A recent study has demonstrated the pivotal involvement of OsWRKY71 in Bph15-mediated resistance (Li et al., 2023). Here, we identified several differentially expressed TFs associated with BPH infestation. Notably, several predominant TF families, including bHLH, MYB, ERF, WRKY, bZIP, NAC, C2H2, TALE, G2-like, HD-ZIP, MYB-related, HSF, and NF-Y, were differentially responsive to BPH infestation. The expression of most TFs was altered in biotype Y BPH-infested rice, indicating their specific roles in defense against the virulent biotype. Notably, the expression of certain other TFs was disturbed specifically in response to biotype 1 BPH infestation. These findings highlight the important functions of TFs in BPH defense and warrant further research to uncover their specific roles.

In conclusion, our study provides valuable insights into the differential defense strategies employed by resistant YHY15 rice (carrying BPH resistance gene Bph15) against avirulent (biotype 1) and virulent (biotype Y) BPH. The BPH defense response was found to involve the modulation of miRNAs, TFs, phytohormone signaling pathways, and the induction of callose deposition. These responses were most noticeable in biotype Y BPH-infested rice plants. These findings contribute to the elucidation of the molecular intricacies underlying rice-BPH interactions and pave the way for further research into the specific genes, pathways, and regulatory elements involved in plant defense against diverse BPH populations. Studying these defense mechanisms will aid our understanding of the intricate interactions between rice and BPH and allow the development of targeted pest control strategies for improved rice cultivation. It is worth noting that the infestation-induced defense responses of YHY15 rice do not appear to alter the survivability of biotype Y BPH, implying the existence of corresponding adaptive responses in the virulent biotype. This result will be the subject of in-depth exploration in future studies.

All raw RNA sequencing data generated in this study have been deposited under the NCBI SRA database under BioProject PRJNA997052 and PRJNA994598.

The manuscript presents research on animals that do not require ethical approval for their study.

BY: Writing – original draft, Writing – review & editing. MG: Investigation, Writing – original draft, Writing – review & editing. YX: Investigation, Writing – review & editing. QY: Writing – review & editing. BL: Formal analysis, Writing – review & editing. ML: Writing – review & editing. YS: Writing – review & editing. CL: Writing – review & editing. JX: Writing – review & editing. JL: Writing – review & editing. WH: Writing – review & editing. HT: Writing – review & editing. PL: Writing – review & editing. QL: Investigation, Supervision, Formal analysis, Writing – review & editing. SJ: Supervision, Project administration, Resources, Funding acquisition, Writing – original draft, Writing – review & editing.