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.