1 INTRODUCTION
Over 200 distinct RNA modifications have been discovered in eukaryotes, of which RNA methylation is a critical post-transcriptional modification that affects gene expression. Among these, the most important and well-studied modification is N6 -methyladenosine RNA methylation (m6A) (Shinde et al., 2023), which refers to the insertion of a methyl substituent on the 6th position N -atom in messenger RNA (mRNA) adenosine (Wei et al., 2018). This occurs widely in eukaryotes such as yeast, fruit flies, plants, and animals (Dominissini et al., 2012) and is the most abundant form of methylation in eukaryotic mRNA and various non-coding RNAs. It controls the fate of RNA at different levels of genetic information transmission, including RNA synthesis and processing, mRNA stability, and translation (Liu et al., 2020b). The m6A modification exerts regulatory functions during RNA synthesis and splicing, influencing the transcription rate, gene stability, and RNA splicing selection. m6A-modified RNA molecules are more prone to recognition and degradation by RNA degradation enzymes. This regulates RNA lifespan and clears abnormal RNA molecules, thus maintaining the dynamic RNA equilibrium within a cell (Sekula et al., 2020). m6A plays a key role in regulating transcription and translation efficiency, thereby controlling the speed of protein synthesis (Bodi et al., 2012; Liu et al., 2020b).
m6A is a reversible chemical modification in which a methyl group is provided by “DONOR,” catalyzed by “WRITER,” removed by “ERASER,” and recognized by the m6A-binding protein “READER” (Shinde et al., 2023).S -adenosylmethionine (SAM) serves as the methyl “DONOR” for almost all cellular methylation reactions (Shen et al., 2016a). “WRITER” is a high-molecular-weight RNA methyltransferase complex capable of writing m6A modifications into mRNA. “ERASER” is usually affected by demethylases; ALKBH9B and ALKBH10B are well-known demethylation proteins in Arabidopsis which can remove m6A from single-stranded RNA of alfalfa mosaic virus (Martínez-Pérez et al., 2017) and Arabidopsis (Duan et al., 2017)in vitro , respectively. The main function of m6A modification depends on its “READER” proteins. In plants, research on m6A “READER” proteins is primarily focused on YTH domain proteins; 13 such proteins have been detected in Arabidopsis , all of which can bind to the m6A position (Wang et al., 2015).
Emergence of various high-throughput sequencing techniques targeting m6A has facilitated functional studies on this RNA modification. These methods include antibody-dependent m6A sequencing and nanopore direct RNA sequencing (DRS) (Wang et al., 2020; Berthelier et al., 2023). Nanopore DRS is a powerful approach that bypasses reverse transcription, requires no amplification, and does not exhibit sequencing bias (Pratanwanich et al., 2021). It can simultaneously detect methylation modification sites on RNA, accurately analyze alternative splicing, and identify novel isoforms (Berthelier et al., 2023). m6A sites are primarily enriched around termination codons and within 3′-untranslated regions (3′-UTRs), exhibiting the m6A consensus motif “RRACH” (R=A/G; H= A/C/U) (Parker et al., 2020). These findings have provided strong evidence for a conserved mechanism of m6A deposition in eukaryotic mRNA.
m6A methylation plays a crucial role in modulating gene expression and biological process in eukaryotes (Wei et al., 2018; Song et al., 2023). In mammalian, different mechanisms of RNA m6A modification in cancer and their potential correlation with cancer prognosis have been elucidated (Wang et al., 2023c). In insects, m6A methylation plays key roles in sex determination, neuronal function, and development (Wang et al., 2021; Chen et al., 2023). Moreover, m6A modification has profound implications in the regulation of pathogen and insecticide resistance. The 5′-UTR of cytochrome P450 gene (CYP4C64 ) in the insecticide-resistant Bemisia tabaci has a m6A mutant site, thus the gene can’t be m6A methylated, thereby increasing gene expression, and enhancing B. tabaciresistance to thiamethoxam (Yang et al., 2021). In plants, m6A modification plays a regulatory role in vegetative growth, floral transition, reproductive development, fruit ripening, photomorphogenesis, and the circadian clock (Tang et al., 2023). m6A also mediates salt tolerance by regulating ROS homeostasis, and auxin signaling in a tissue-specific manner (Wang et al., 2022). In addition, m6A methylation is increased in rice infected with rice stripe virus (RSV) or rice black-stripe dwarf virus (RBSDV), several antiviral pathway-related genes—such as RNA silencing, resistance, and fundamental antiviral phytohormone metabolism-related genes—are methylated by m6A (Zhang et al., 2021a). m6A modification might be an epigenetic mechanism that regulates RBSDV replication in small brown planthoppers (SBPH) and maintains a certain viral threshold required for persistent transmission (Tian et al., 2021). Thus, the modification of m6A in plants may also play an important role in regulating plant defense against insect, but this has rarely been explored to date.
When attacked by herbivores, plants activate early signaling events, such as mitogen-activated protein kinases (MAPKs). Then the production of defense-related phytohormones, such as jasmonic acid (JA) and salicylic acid (SA), are induced, which are well known to regulate the production of defensive compounds and thus confer resistance to (Erb et al., 2019). The brown planthopper (BPH; Nilaparvata lugens Stål) is a monophagous sap-sucking herbivore that causes severe yield reductions and economic losses in rice crops (Otuka, 2013). It causes direct damage to rice plants by feeding on phloem sap via its ovipositor and laying egg clusters in tissues (Bass et al., 2011). The JA upregulates sakuranetin synthesis in rice and enhances resistance against BPH (Liu et al., 2023). While SA mediates the accumulation of anti-insect callose in the phloem (Wang et al., 2023b). To date, numerous BPH resistance genes (Bphs ) have been well-documented in rice. Among them, several Bphs regulate phytohormones signaling pathways and exhibit various mechanisms of insect resistance (Hu et al., 2011; Li et al., 2023; Pannak et al., 2023). Bph14 activates SA-mediated callose deposition in rice leaf sheath and exhibits BPH resistance in early stage rice seedlings (Du et al., 2009). For BPH, successful phloem feeding is achieved by penetrating the sclerenchyma tissue of the rice epidermis using its stylet (Shi et al., 2021). The sclerenchyma tissue is mainly composed of cellulose, hemicellulose, and lignin, providing mechanical strength and stability to rice stems. Bph30 andBph40 were highly expressed in sclerenchyma cells and enhanced cellulose and hemicellulose synthesis, which makes the cell walls stiffer and sclerenchyma thicker and thus enhance resistance to BPH by inhibiting insect feeding (Shi et al., 2021). Upon BPH infestation, rice defense is activated but growth is suppressed (Jin et al., 2023). The crosstalk of defense- and growth-related phytohormones plays an important role in the growth–defense trade-offs (Li et al., 2015). JA signaling activates defense responses and plays a central role in prioritizing defense over growth during herbivore attacks, by suppressing growth-related phytohormones pathways, such as auxin and GA (Hou et al., 2010; Chen et al., 2011; Yang et al., 2012; Jin et al., 2023).
Using nanopore DRS approach combined with RNA sequencing, we aimed to examine the interactions between rice and BPH by investigating the dynamic modulation of m6A modification in rice genome. We identified the specific genes and pathways that are influenced by these modifications, to deepen our understanding of how m6A modifications contribute to rice defenses against BPH at the expense of plant growth.