Abstract

   Rice (Oryza sativa L.) serves as a substitute for bread and is a staple
   food for half of the world’s population, but it is heavily affected by
   insect pests. The fall armyworm (Spodoptera frugiperda) is a highly
   destructive pest, threatening rice and other crops in tropical regions.
   Despite its significance, little is known about the molecular
   mechanisms underlying rice’s response to fall armyworm infestation. In
   this study, we used transcriptome analysis to explore the global
   changes in gene expression in rice leaves during a 1 h and 12 h fall
   armyworm feeding. The results reveal 2695 and 6264 differentially
   expressed genes (DEGs) at 1 and 12 h post-infestation, respectively.
   Gene Ontology (GO) and KEGG enrichment analyses provide insights into
   biological processes and pathways affected by fall armyworm feeding.
   Key genes associated with hormone regulation, defense metabolic
   pathways, and antioxidant and detoxification processes were
   upregulated, suggesting the involvement of jasmonic acid (JA)
   signaling, salicylic acid biosynthesis pathways, auxin response, and
   heat shock proteins in defense during 1 h and 12 h after fall armyworm
   infestation. Similarly, key genes involved in transcriptional
   regulation and defense mechanisms reveal the activation of calmodulins,
   transcription factors (TFs), and genes related to secondary metabolite
   biosynthesis. Additionally, MYB, WRKY, and ethylene-responsive factors
   (ERFs) are identified as crucial TF families in rice’s defense
   response. This study provides a comprehensive understanding of the
   molecular dynamics in rice responding to fall armyworm infestation,
   offering valuable insights for developing pest-resistant rice varieties
   and enhancing global food security. The identified genes and pathways
   provide an extensive array of genomic resources that can be used for
   further genetic investigation into rice herbivore resistance. This also
   suggests that rice plants may have evolved strategies against
   herbivorous insects. It also lays the groundwork for novel
   pest-resistance techniques for rice.

   Keywords: rice plant defense, transcriptome analysis, plant–insect
   interaction, secondary metabolism, signaling pathways, stress response

1. Introduction

   Herbivore attacks significantly reduce plant growth and crop
   productivity. Host plants have evolved numerous defense mechanisms to
   protect themselves against herbivorous challenges as a result of
   coevolution and interactions between plants and herbivores [[28]1].
   Constitutive defenses for herbivores are the primary expression of
   physical and chemical defensive traits in the absence of herbivores,
   whereas induced defenses are triggered only after herbivore attack
   [[29]2,[30]3]. Rice (Oryza sativa) and its insect pests have long been
   used as model systems to investigate the intricate relationships of
   plant–insect interactions [[31]4,[32]5]. Moreover, rice (Oryza sativa
   L.) serves as a crucial alternative to wheat for bread production and
   is the primary staple food for nearly half of the global population.
   However, rice cultivation faces significant challenges due to the
   severe impact of insect pests, which cause substantial yield losses and
   threaten food security in many rice-producing regions [[33]6,[34]7].
   Despite the increasing demand, research shows that pests and crop
   diseases cause 20–40% of global food production losses, with pests
   directly accountable for or contributing to a substantial percentage of
   these losses [[35]1]. Among the numerous insect pests of rice, the fall
   armyworm (FAW), Spodoptera frugiperda, is a highly destructive crop
   pest that is native to the tropical and subtropical regions of the
   American continent [[36]8]. It poses a significant threat to tropical
   annual crops due to its ability to feed on various range of host
   plants, including rice [[37]9,[38]10]. FAW is composed of two
   genetically distinct strains, the rice (R-strain) and corn (C-strain)
   strains, which have been extensively studied [[39]11,[40]12].
   Understanding the molecular basis of host defense mechanisms against
   insect attack is crucial for effective insect pest management in crop
   production.

   Plant–insect interaction is an intricate evolutionary system defined by
   a continuing arms race that forces both organisms to constantly
   coevolve numerous defense mechanisms and find strategies for their
   survival [[41]13]. This poses serious problems for global agriculture
   productivity and food security [[42]14]. This selection process of host
   plants is intricate and has evolved over millions of years of
   coevolution between these insects and plants [[43]15]. The act of
   insects feeding on plants stimulates the generation of various physical
   and chemical defenses in plants. Plants can activate specific defense
   mechanisms in response to both mechanical damage and cues from insects
   [[44]16]. These defenses can be harmful, making the plant toxic, or
   reducing the digestibility of food for the insect, as well as
   attracting natural enemies of the insect [[45]17]. Numerous studies
   have demonstrated that plants’ resistance and defensive responses to
   insect attacks involve intricate signaling pathways. These pathways are
   capable of recognizing specific effector molecules present in insect
   saliva. Upon recognition, these pathways activate robust resistance
   mechanisms within the host plants, enabling them to effectively defend
   against insect herbivory [[46]18,[47]19,[48]20]. Activating defense
   reactions is dependent on immediately and accurately identifying
   chemical patterns associated with herbivores. Early signaling events
   are crucial to the basic plant response to herbivory because they
   initiate subsequent signaling transduction pathways
   [[49]21,[50]22,[51]23]. Host plant resistance should be considered one
   of the important ways to reduce crop losses.

   Induced defense mechanisms are the results of intricately synchronized
   sequential changes at the cellular level, changes that activate several
   signaling pathways [[52]24]. These first processes further activate the
   calcium flux, the production of reactive oxygen species, membrane
   potential depolarization, and mitogen-activated protein kinase (MAPK)
   [[53]23,[54]25]. The plant hormonal signaling network links sensing and
   early signaling events to wide transcriptional reorganization and
   defense induction in the dynamic defense process [[55]26,[56]27]. This
   complex sequence triggers the production of defensive chemicals,
   proteins, and secondary metabolites, resulting in phenotypic
   alterations in plants [[57]26,[58]28]. The signaling molecules initiate
   defense responses by generating secondary metabolites, which can
   include toxic compounds that contribute to direct insecticidal defense
   [[59]29,[60]30]. Previous studies reported that secondary metabolites,
   which include nitrogen- and sulfur-containing chemicals, terpenoids,
   and phenolics, as well as plant defensive proteins, are inducible
   defenses against herbivores that are linked to the activation of
   defense systems in plants [[61]31,[62]32]. Plant hormones that are
   involved in herbivore-induced defense signaling include auxin,
   gibberellins, ethylene (ET), jasmonic acid (JA), salicylic acid (SA),
   cytokinins, and abscisic acid (ABA) [[63]33]. The most studied of these
   phytohormone pathways are JA and SA [[64]21,[65]23,[66]34]. While the
   SA route is mostly activated by piercing–sucking herbivores, the JA
   pathway is normally activated in response to chewing insects and
   cell-content feeders [[67]35]. In dicotyledonous plants, the JA pathway
   is widely recognized as the principal and conserved mechanism
   regulating plant resistance to various kinds of insects
   [[68]25,[69]26,[70]36]. However, whether and how plant hormones can
   regulate herbivore-induced defense in rice after fall armyworm
   infestation is unclear.

   However, numerous studies have used a next-generation sequencing
   approach to examine the interactions between phytophagous insects and
   their host plants [[71]37,[72]38]. Conducting impartial analyses of the
   transcriptional profile resulting from RNA sequencing (RNA-Seq) is an
   effective approach to investigating host plant adaptation mechanisms,
   and it enables the functional characterization of differentially
   regulated genes under varying conditions. To profile the transcripts
   expressed in the rice plants 0 h (control), 1 h, and 12 h after
   infestation by fall armyworm larvae, we used transcriptome sequence
   analysis to identify the expression level of genes. Several genes that
   were either commonly or exclusively expressed, upregulated or
   downregulated, were identified in rice plants after fall armyworm
   infestation. Some of these genes are associated with different
   metabolic processes that produce substances such as SA, JA, ET, and
   other secondary metabolites. Using two high-throughput sequencing
   methods, our results provide a comprehensive and preliminary
   understanding of the changes in the rice plant transcriptome during
   insect infestation. The candidate genes that have been identified in
   the current study are important starting points for further
   investigation into the functions of key genes involved in rice
   plant–insect interactions.

2. Results

2.1. Overview of Quality Control and Mapping Statistics of Transcriptome
Dataset

   The FAW larvae caused damage to rice leaves during the 12 h feeding
   session ([73]Figure 1). To examine the global transcriptome changes
   resulting from this feeding, an mRNA analysis was carried out on nine
   libraries, each of which represented unique time intervals (0, 1, and
   12 h). Together, these libraries included three biological replicates
   at every sample interval. For all samples (Control, Rice_1H, and
   Rice_12H), there was at least 4.9-to-6.8 GB of data with Q20 and Q30
   quality scores higher than 97% ([74]Table 1). In total, 53.16–53.71% of
   the reads were GC-containing, and 44.84–56.19 million of those reads
   were uniquely mapped to the rice IRGSP–1.0 reference genome. The range
   of unique mapping rates was from 89.73% to 91.36% ([75]Table 1). Based
   on our analyses of the gene structure, most mapped reads (92.40–95.70%)
   were found to be in exons. The uniquely mapped reads were used in
   further investigations.

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Showing the layout of experimental design: (A) infestation of rice
   plants with FAW larvae for 1 h and 12 h and control; (B) larvae were
   collected from infested host plants for RNA extraction and cDNA
   synthesis; (C) high-throughput sequencing and raw data; (D) biological
   insights; and (E) RTq-PCR analysis for the verification of DEGs data
   obtained from transcriptome analysis.

Table 1.

   Summary of the sequencing data.
   Sample Raw Data Valid Data Valid Ratio (Reads) Q20% Q30% GC Content%
   Read Base Read Base
   Control1 52,435,330 7.87G 46,488,478 6.97G 88.66 99.96 97.85 49.5
   Control2 49,733,028 7.46G 42,199,050 6.33G 84.85 99.96 97.96 49
   Control3 48,440,398 7.27G 40,780,310 6.12G 84.19 99.95 97.94 49
   Rice_12H1 39,611,302 5.94G 38,707,090 5.81G 97.72 99.97 96.39 50
   Rice_12H2 36,895,594 5.53G 35,867,176 5.38G 97.21 99.97 96.16 49.5
   Rice_12H3 47,139,810 7.07G 37,954,992 5.69G 80.52 99.96 97.89 50
   Rice_1H1 44,931,270 6.74G 43,857,698 6.58G 97.61 99.97 96.94 52.5
   Rice_1H2 33,816,536 5.07G 32,920,526 4.94G 97.35 99.97 96.24 50
   Rice_1H3 37,937,056 5.69G 35,665,200 5.35G 94.01 99.97 96.45 48
   [78]Open in a new tab

2.2. Analyses of Differentially Expressed Genes

   DESeq2 software version: 1.45.3 was used for determining the
   upregulation and downregulation of genes based upon relative Fragments
   Per Kilobase Million (FPKM) counts (x-axis: log2fold change; y-axis:
   −log (p-value) [79]Figure 2. A total of 2695 and 6264 differentially
   expressed genes (DEGs) were regulated in rice leaves at various time
   points (Rice-1h vs. control and Rice-12h vs. control) after FAW larvae
   infestation ([80]Figure 3). Among them, there were 1716 upregulated and
   979 downregulated genes in Rice-1h vs. control and 3134 upregulated and
   3120 downregulated genes in Rice-12h vs. control after FAW larvae
   infestation ([81]Figure 3).

Figure 2.

   [82]Figure 2
   [83]Open in a new tab

   Bar graph showing a total number of up- and downregulated
   differentially expressed genes (DEGs) in transcriptome analysis of
   Rice-1h vs. control and Rice-12h vs. control after FAW larvae
   infestation.

2.3. GO Enrichment Analysis of DEGs Induced by FAW Larval Feeding

   A Gene Ontology (GO) enrichment analysis was conducted to categorize
   the functional roles of differentially expressed genes (DEGs) in rice
   plants subjected to feeding by fall armyworm (FAW). This analysis aimed
   to identify biological processes, molecular functions, and cellular
   components associated with DEGs. The study specifically examined genes
   that exhibited a differential expression at both an early time point (1
   h after FAW feeding compared to the control) and a late time point (12
   h after FAW feeding compared to the control) ([84]Figure 3A,B). The
   results show the upregulated genes associated with the following at one
   hour: “cellular response to nucleus”, “regulation of jasmonic
   acid-mediated signaling pathway”, “response to acid chemical”, and
   “regulation of defense response”. Upregulated differentially expressed
   genes (DEGs) showed notable enrichment in several Gene Ontology (GO)
   biological process, and these comprised procedures like “biological
   process”, “regulation of transcription DNA”, “defense response”,
   “protein phosphoration”, and “abscisic acid activated signaling
   pathways”. Interestingly, “DNA binding transcription factor”,
   “oxidoreductase activity”, “peroxidase activity”, and “kinase activity”
   showed notable enrichment in the DEGs at different time points
   ([85]Figure 4A,B).

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   Gene Ontology (GO)-enriched terms of differentially expressed genes
   (DEGs) and unigenes in Rice-1h vs. control (A) and Rice-12h vs. control
   (B) after FAW larvae infestation. The x-axis lists the sub-GO terms
   under categories of biological process, cellular component, and
   molecular function. The y-axis is the number of DEGs involved in each
   term.

Figure 3.

   [88]Figure 3
   [89]Open in a new tab

   Gene Ontology (GO) classification of transcripts. The number of
   significantly up- and downregulated unigenes in Rice-1h vs. control (A)
   and Rice-12h vs. control (B) after FAW larvae infestation.

2.4. KEGG Enrichment Pathways Analysis of DEGs Induced by FAW Larval Feeding

   In this study, the genes that were regulated at one hour and twelve
   hours both showed a notable enrichment in plant hormone signal
   transduction, according to the KEGG enrichment analysis. For the
   Rice-1h vs. control mark, the regulation of genes displayed enrichment
   in terms of the regulation of defense response ([90]Figure 5A,B),
   whereas the regulated genes were significantly enriched in pathways
   linked to the “response to wounding”, regulation of defense response
   and secondary metabolite biosynthetic process, and regulation of
   jasmonic acid-mediated signaling pathways in Rice-12h vs. control after
   FAW larvae infestation ([91]Figure 5A,B). The overall results of the
   FPKM cluster analysis ([92]Figure 6A,B) of differentially expressed
   genes were clustered using their log2 (FPKM + 1) value in the form of a
   heat map ([93]Figure 6A,B). The clustering of genes with similar
   expression levels was carried out to better understand gene expression
   in relation to their biological relevance.

Figure 5.

   [94]Figure 5
   [95]Open in a new tab

   Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and pathways
   of in Rice-1h vs. control (A) and Rice-12h vs. control (B) after FAW
   larvae infestation.

Figure 6.

   [96]Figure 6
   [97]Open in a new tab

   The expression patterns of defensive–responsive genes in in Rice-1h vs.
   control (A) and Rice-12h vs. control (B) after FAW larvae infestation.
   Hierarchical clustering heat map depicting overall results of the FPKM
   clustering using log2 (FPKM + 1) values. The red and blue squares
   indicate genes with high or low gene expression levels, respectively.

2.5. Identification of Important Genes Based on Functional Classification and
GO Enrichment Analysis of DEGs in Response to FAW Larvae Feeding

   Plant responses to insect herbivores depend heavily on early signaling
   processes, such as the proteins that control the expression of target
   genes, such as transcription factors (TFs) and transcriptional
   regulators (TRs). The activation of these signals in response to stress
   mediated by insects can provide important information about the
   molecular mechanisms that underlie plant responses to stress caused by
   insect herbivory. In our study, auxin-responsive protein IAA2 and LRR
   receptor-like serine/threonine-protein kinase in Rice-1h vs. control
   and Rice-12h vs. control, were among the differentially expressed genes
   (DEGs) ([98]Supplementary Table S1). Notably, the families with the
   highest number of linked TFs or TRs included ethylene-responsive
   transcription factor, anthocyanidin reductase, zinc finger A20 and AN1
   domain-containing stress-associated protein, basic helix–loop–helix
   (bHLH) genes, and WRKY genes in Rice-1h vs. control and Rice-12h vs.
   control ([99]Supplementary Table S2).

2.6. Identification of Genes Related to Secondary Metabolism Transcriptional
Regulation, Hormone Regulation, and Antioxidant and Detoxification Processes

   A further analysis indicated that MYB, WRKY, ethylene responsive
   factors (ERFs), zinc finger domain proteins, and basic helix–loop–helix
   (bHLH) are the TF families that are widely reported to be involved in
   plant defense responses. In this study, we identified MYB (5,9), WRKY
   (9,13), ethylene responsive factors (ERFs) (19,24), zinc finger domain
   proteins (16,30), and basic helix–loop–helix (bHLH) (12,25), which were
   upregulated in Rice-1h vs. control genes) and Rice-12h vs. control
   after FAW infestation. ([100]Figure 7A,B) ([101]Supplementary Table
   S3). Similarly, a chalcone synthase family protein is a key enzyme
   involved in the biosynthesis of flavonoids to encode chalcone synthase
   (CHS) and is required for the accumulation of purple anthocyanins in
   leaves and stems.

Figure 7.

   [102]Figure 7
   [103]Open in a new tab

   A number of upregulated genes related to secondary metabolism (A) and
   transcriptional regulation in Rice-1h vs. control (A) and Rice-12h vs.
   control (B) after FAW larvae infestation.

   In this transcriptome study, the results showed that 16 genes in
   Rice-1h vs. control and 23 in Rice-12h vs. control related to the
   metabolism of phenylpropanoid biosynthesis, which is central to
   producing defense-related compounds, were upregulated ([104]Figure
   8A,B). Similarly, further results indicated that five and eight genes
   related to the flavanone 3-dioxygenase were upregulated in Rice-12h vs.
   control and in Rice-12h vs. control respectively ([105]Supplementary
   Table S3). Furthermore, key regulatory genes belonging to the subgroup
   of late anthocyanin biosynthesis genes were significantly upregulated
   in genes in Rice-1h vs. control (7 genes) and Rice-12h vs. control (11)
   after FAW infestation. Additionally, the involvement of Cytochrome P450
   (CYP) genes was observed in Rice-1h vs. control (17 genes) and Rice-12h
   vs. control (53 genes) after FAW infestation. Furthermore, genes
   related to secondary metabolism, such as those involved in the
   tetracyclic triterpenoid biosynthetic process and DIMBOA
   UDP-glucosyltransferase BX8, exhibited higher transcript abundance in
   Rice-1h vs. control and Rice-12h vs. control following FAW infestation,
   specifically related to steroid and zeatin biosynthesis.

Figure 8.

   [106]Figure 8
   [107]Open in a new tab

   A number of upregulated genes related to hormone regulation (A) and
   antioxidant and detoxification processes in Rice-1h vs. control (A) and
   Rice-12h vs. control (B) after FAW larvae infestation.

   In addition, lipoxygenase 2 LOX2 (5,6), a JA-signaling and -biogenesis
   gene (11,16), was detected as an upregulated gene in Rice-1h vs.
   control genes) and Rice-12h vs. control after FAW infestation
   ([108]Figure 9 and [109]Supplementary Table S4). Upregulation of allene
   oxide synthase (AOS) (3,5), is needed for JA production in Rice-1h vs.
   control genes and Rice-12h vs. control after FAW infestation. In
   addition, the upregulation expression of auxin-responsive protein
   related to signaling pathway, and Auxin response factor ARF (2,2), were
   upregulated in Rice-1h vs. control genes and Rice-12h vs. control after
   FAW infestation respectively. Similarly, we found disease resistance
   protein (9,8) which showed higher transcript abundance genes in Rice-1h
   and Rice-12h vs. control after FAW larvae infestation. Similarly,
   leucine-rich repeat (LRR) (15,15) protein kinase was highly expressed
   in Rice-1h vs. control genes and Rice-12h vs. control. In addition,
   genes encoding the cysteine-rich like kinase (7,2) and receptor-like
   proteins (RLPs) (5,11) were highly expressed upon FAW larvae feeding.
   Similarly, we identified the heat shock protein HSP 70 (4,8), which was
   present as an upregulated expression in Rice-1h vs. control genes and
   Rice-12h vs. control after FAW larvae feeding, highlighting the
   function of HSPs in plant defense against pathogenic infection and
   reduced accumulation of ROS. A further analysis indicated that, among
   detoxification genes, UDP-glycosyltransferase (23,31) significantly
   upregulated in Rice-1h vs. control genes and Rice-12h vs. control after
   FAW larvae feeding, suggesting this gene may play an important role in
   the rice resistance to FAW larvae feeding ([110]Figure 8A,B and
   [111]Supplementary Table S4). The results of the FPKM cluster analysis
   of differentially expressed genes related to secondary metabolism,
   transcriptional, hormones regulation, and antioxidant and
   detoxification processes in Rice-1h vs. control and Rice-12h vs.
   control after FAW larvae infestation were clustered using their log2
   (FPKM + 1) value in the form of a heat map ([112]Supplementary Figure
   S1A,B).

2.7. qRT-PCR for the Validation of Differentially Expressed Genes (DEGs)

   We selected twenty differentially expressed genes (DEGs) for a
   quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
   to verify the gene expression results obtained from the transcriptome
   data ([113]Figure 9A–F). These selected genes include those that encode
   secondary metabolism, transcriptional regulation, hormone regulation,
   and antioxidant and detoxification processes ([114]Figure 10A–H). In
   addition, our qRT-PCR results showed similar relative expression
   profiles of the selected DEGs with the RNA-Seq results in Rice-1h and
   Rice-12h vs. control after FAW larvae infestation.

Figure 10.

   [115]Figure 10
   [116]Open in a new tab

   (A–H) Results for the qRT-PCR confirmation of the DEGs library and
   qRT-PCR analysis of upregulated genes of antioxidants and
   detoxification genes: Hormone regulation (A–D). Antioxidant and
   detoxification processes (E–H) in Rice-1h vs. control and Rice-12h vs.
   control after FAW larvae infestation.

Figure 9.

   [117]Figure 9
   [118]Open in a new tab

   (A–F). Results for the qRT-PCR confirmation of the DEGs library.
   qRT-PCR analysis of ten upregulated genes of detoxification genes:
   transcriptional regulation (A–F) and hormone regulation (G–L) in
   Rice-1h vs. control and Rice-12h vs. control after FAW larvae
   infestation. Different letters a and b indicate significant
   differences.

3. Discussion

   There are always potential risks to plants from a wide variety of
   herbivores. Plants have evolved defense mechanisms that are activated
   in response to insect herbivory to protect themselves against insects.
   Direct defenses, such as the activation of peroxidase, polyphenol
   oxidase, and proteinase inhibitor, are among these mechanisms
   [[119]39,[120]40]. Indirect defenses also function, such as the
   emission of a mixture of volatile fatty acid derivatives,
   phenylpropanoid chemicals, and terpenoids [[121]41]. In this study, the
   transcriptomic analysis results revealed the differential expression of
   defense-related genes and pathways when rice plants were infested with
   FAW larvae for 1 h and 12 h. Our results are consistent with those of a
   previous study in which defense-related genes and pathways were
   significantly upregulated when plants were infested with herbivores
   [[122]28,[123]42,[124]43]. Similarly, the results showed that genes
   related to the metabolism of phenylpropanoid biosynthesis, and
   flavanone 3-dioxygenase were significantly upregulated in rice after 1
   h and 12 h of infestation, as compared to the control. Previous studies
   have reported the metabolism of phenylpropanoid, a central to producing
   defense-related compounds, including anthocyanins and flavonoids in
   plants [[125]44,[126]45,[127]46]. Transcription factors, including
   MYBs, facilitate the activation of this critical regulatory gene, which
   is part of the late anthocyanin biosynthesis genes subgroup
   [[128]47,[129]48]. Additionally, the possible involvement of Cytochrome
   P450 (CYP) genes was found in Rice-1h vs. control and Rice-12h vs.
   control after FAW infestation. Consistent with our results, a previous
   investigation suggests that Cytochrome P450 (CYP) genes may play a role
   in plant defense [[130]49,[131]50]. Our results concluded that the
   plant immune system plays a crucial role in triggering induced defenses
   against herbivores.

   Furthermore, the involvement of the TF families was found in our study
   in plant defense systems and has also been well-documented in previous
   studies [[132]51]. MYB, WRKY, zinc finger domain proteins, ethylene
   response factors (ERFs), and basic helix–loop–helix (bHLH) are among
   these families [[133]52]. Similarly, by encoding chalcone synthase
   (CHS), transparent testa4 (TT4), a member of the chalcone synthase
   family proteins, is a crucial enzyme in the synthesis of flavonoids
   [[134]53]. It is important because it promotes the development of
   purple anthocyanins in stems and leaves [[135]54,[136]55]. Numerous
   studies have shown that the expression of genes linked to anthocyanin
   biosynthesis is regulated by a conserved regulatory complex called
   MYB-bHLH-WD40 (MBW) [[137]56,[138]57]. Additionally, after fall
   armyworm (FAW) infestation, we found a substantial upregulation of WRKY
   genes in rice plants. In previous studies, it has been reported that
   OsWRKY70 and JA are part of the same signaling cascade and are positive
   regulators of rice resistance to chewing herbivores [[139]58,[140]59].
   The expression levels of zinc finger proteins of the Cys2/His2-type and
   C2H2-type were upregulated in rice when infested by FAW larvae.
   According to Han et al. [[141]60], Cys2/His2-type zinc finger proteins
   improve resistance against insect pests, in addition to being involved
   in plant stress responses. Furthermore, after infestation by FAW
   larvae, the rice plants exhibited activation of ERFs, a critical subset
   of transcription factors involved in integrating ethylene (ET) and
   jasmonic acid (JA) signals [[142]61,[143]62,[144]63]. In addition,
   previous studies have reported the roles of genes related to
   phytohormone signaling pathways, including jasmonic acid (JA),
   salicylic acid (SA), abscisic acid (ABA), and ethylene (ET), which
   result in the production of defensive compounds in plants in response
   to herbivores, including FAW [[145]64,[146]65,[147]66]. In this
   experiment, transcript abundances of ERFs were increased in rice
   infested with FAW larvae as compared to the control. However,
   Arabidopsis has demonstrated the function of ERF1 as a regulator of
   ethylene responses following insect attack [[148]67]. This indicates
   that rice plants may have developed specific defense mechanisms over
   time to counteract herbivorous insect attacks. These strategies could
   have evolved as an adaptive response to the pressures exerted by insect
   herbivory, allowing rice plants to enhance their survival and
   reproductive success in environments where insect pests pose a
   significant threat.

   In this study, lipoxygenase 2 LOX2, a JA-signaling and -biogenesis
   genes, was detected as an upregulated gene in rice-1h vs. control genes
   and Rice-12h vs. control after FAW infestation. Previous studies showed
   that JA signaling has closely been associated with defense mechanisms
   against pathogens and insects [[149]33,[150]68,[151]69]. A previous
   study reported that damage caused by fall armyworms in rice plants
   triggers a substantial early response linked to hormones such as
   jasmonates [[152]30]. Similarly, upregulation of allene oxide synthase
   (AOS), needed for JA production, in rice plants after FAW infestation
   suggests that rice resistance to the insect attack is enhanced by the
   activation of JA signaling pathways [[153]28]. Our results agree with
   the previous studies where the expression levels of LOX and AOS
   significantly increased after infestation [[154]69,[155]70]. In
   addition, the upregulation expression of auxin-responsive protein
   related to the signaling pathway, and auxin response factor ARF were
   upregulated in Rice after FAW larvae infestation. SAUR genes are
   related to cell division [[156]71,[157]72] and are reportedly regulated
   by the auxin level, indicating that this process could be impaired by
   FAW larvae feeding. In this experiment, we found disease-resistance
   protein which showed higher transcript abundance in rice after FAW
   larvae infestation. Similarly, leucine-rich repeat (LRR) protein kinase
   was highly expressed in rice after FAW larvae feeding, as compared to
   the control. A previous study showed that treatment with OS of
   Spodoptera frugiperda increased the expression level of OsLRR-RLK1, JA,
   SA, and ethylene biosynthesis genes and the activity of trypsin
   protease inhibitor (TrypPI) in rice [[158]73]. In addition, genes
   encoding the Cysteine-rich like kinase and receptor-like proteins
   (RLPs) were highly expressed upon FAW larvae feeding. This highly
   upregulated CRK gene seems to indicate its potential role in resistance
   against insect pests [[159]74]. We also observed receptor-like proteins
   (RLPs) and receptor-like kinase (RLK) genes that have a direct effect
   on the insect and pathogen [[160]75,[161]76]. Similarly, we identified
   the heat shock protein HSP 70 and detoxification genes like
   UDP-glycosyltransferase significantly upregulated in rice as compared
   to the control after FAW larvae feeding. Heat shock proteins (HSPs) and
   reactive oxygen species (ROS) are essential for promoting ROS
   scavenging activity and improving stress tolerance ([162]Figure 11)
   [[163]77,[164]78]. Our study about the role of UDP-glycosyltransferase
   is further supported by earlier studies on spider mites in resistant
   and susceptible common bean cultivars [[165]43]. In addition, our
   qRT-PCR results showed similar relative expression profiles of the
   selected DEGs with the RNA-Seq results in Rice-1h vs. control and
   Rice-12h vs. control after FAW larvae infestation. This study showed
   consistency with other study findings that show similar fold changes
   between qPCR and RNA-Seq data.

Figure 11.

   [166]Figure 11
   [167]Open in a new tab

   A working model diagram of molecular insights into the rice plant
   defense mechanism in response to an insect-attacked plant.
   Insect-derived elicitors are perceived by unidentified receptors on the
   plasma membranes, triggering rapid activation of MAPKs followed by
   biosynthesis of phytohormones, JA, JA-Ile, and ethylene. After several
   steps of signaling transduction, transcription factors (MYC2 and ERFs,
   for instance) regulate the accumulation of non-volatile secondary
   metabolites (such as TPIs in rice), which function as direct defenses
   against herbivores.

   In conclusion, our study unveils the intricate defense mechanisms
   triggered by rice plants in response to an infestation of fall armyworm
   (FAW) larvae. Significant activation of defense-related genes and
   pathways was revealed by transcriptomic analysis in rice after FAW
   larvae infestation. Important transcription factors, including WRKY,
   MYB, and ERFs, as well as genes involved in the synthesis of flavonoids
   and phenylpropanoid, are crucial. Transcriptomic results showed that
   the plants’ defense mechanisms are further activated by the
   overexpression of auxin response genes and JA signaling pathways. The
   study is noteworthy for identifying particular genes, such as
   detoxification genes, RLPs, RLKs, and CRK, and highlighting their
   possible functions in increasing resistance against FAW larvae. Current
   research broadens our understanding of the intricate relationship
   between plants and herbivores, offering insights into the development
   of effective strategies in crop protection. Further research is needed
   to explore whether modulating these key genes impacts the performance
   of FAW larvae feeding on rice and to gain a deeper understanding of the
   efficiency of rice immunity against herbivores.

4. Materials and Methods

   Larval populations of S. frugiperda were initially collected from two
   different corn fields located in Haikou, China. These larvae were
   subsequently placed alongside maize plants in a controlled environment
   with a temperature of 25 ± 2 °C and a 14:10 h light/ dark photoperiod.
   The plants were grown in a controlled plant growth chamber at 28 ± 2 °C
   with a 14:10 h light/dark photoperiod at the Ministry of Education Key
   Laboratory for Ecology of Tropical Islands, College of Life Sciences,
   Hainan Normal University, Haikou, China). The newly emerging moths were
   paired and placed in mating cages once they reached the pupation stage.
   There, they were fed a 10% honey solution. The population was used in
   host selection tests after undergoing a generation cycle on
   one-month-old maize seedlings for three generations before
   investigation.

4.1. Plants and Insect Infestation

   The seeds of the commercial rice plant Oryza sativa “White Rice”
   (Báifàn) were purchased from China and planted in vermiculite mixed
   with peat moss (4:1) in plastic trays. The rice plants were grown in a
   controlled plant growth chamber at 28 ± 2 °C with a 14:10 h light/dark
   photoperiod at the Ministry of Education Key Laboratory for Ecology of
   Tropical Islands, College of Life Sciences, Hainan Normal University,
   Haikou, China. Thirty-day-old plants were used for experiments. After 3
   generations were reared in the laboratory, a total of 90 third-instar
   larvae were released in plastic pots, 20 cm in height and 15 cm in
   width, containing 30-day-old rice plants. A total of three treatments
   with three replicates of each treatment contained 10 larvae for each
   replicate (each individual plant was treated as a single replicate).
   Plastic pots, 20 cm in height and 15 cm in width, containing 30-day-old
   rice plants without FAW larvae served as a control treatment. Damaged
   leaves of rice plants were collected after infestations with larvae
   after one hour and twelve hours ([168]Figure 1). The treated leaves
   were then quickly frozen with liquid nitrogen and kept at −80 °C until
   the RNA isolation.

4.2. RNA Extraction, Library Preparation, and RNA-Sequencing Analysis

   Total RNA was isolated (using the TRIzol1 Reagent (Invitrogen, Waltham,
   MA, USA) [169]https://www.thermofisher.com/cn/en (accessed on 26 July
   2023) by the manufacturer’s instructions) from three biological
   replicates, each made up of combined samples from at least three
   different plants. The RNA samples were then subjected to an RNase-free
   DNaseI treatment (Invitrogen; [170]https://www.thermofisher.com)
   (accessed on 13 September 2022). Using a NanodropTM 2000
   spectrophotometer, the quantity and quality of the RNAs were evaluated.
   All RNA samples were transferred to the Beijing Genomic Institute (BGI)
   in China for transcriptome sequencing and library preparation to
   guarantee proper analysis. Paired-end (2 × 150 bp) readings were
   produced using the Illumina HiSeq 2000 platform.

4.3. Reads’ Preprocessing and Differential Expression Analysis

   The initial raw reads, obtained in Fastq format from the BGI institute
   website, underwent preprocessing and differential expression analysis.
   Trimmomatic v0.39 software [[171]79] was utilized for quality control
   (QC), trimming low-quality reads, adapters, and Illumina-specific
   sequences. Quality thresholds of a minimum length of 50 bp and a
   minimum quality score of 30 were applied. Evaluation of raw sequence
   quality, both before and after filtering, was conducted using FastQC
   ([172]https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
   (accessed on 26 July 2023)). Clean reads were aligned to the reference
   genome of rice using the RNA-Seq aligner STAR software version 2.7.11.
   Genes differential expression analysis was performed by DESeq2 software
   between two different groups (and by edgeR between two samples). The
   genes with the parameter of false discovery rate (FDR) below 0.05 and
   absolute fold change ≥2 were considered differentially expressed genes.
   Differentially expressed genes were then subjected to enrichment
   analysis of GO functions and KEGG pathways [[173]80,[174]81].

4.4. GO Terms and KEGG Pathways Enrichment Analysis

   Through the use of the AgriGO module and Singular Enrichment Analysis
   (SEA), the roles of the DEGs were made clear. Finding enhanced Gene
   Ontology terms from the agriGO database [[175]82] was the aim of this
   research, which had a significance threshold set at 0.05. Additionally,
   using the KOBAS 3.0 web server ([176]http://kobas.cbi.pku.edu.cn/
   (accessed on 26 July 2023)), an enrichment analysis of Kyoto
   Encyclopedia of Genes and Genomics (KEGG) pathways for the DEGs was
   conducted. KEGG pathways were deemed significantly enriched if their
   adjusted p-value was less than 0.05.

4.5. Validation of Transcripts DEGs Using qRT-PCR

   For the validation of candidate DEGs, qRT-PCR was conducted for six
   selected DEGs, with Actin serving as the reference gene. Primers were
   designed using Geneious ([177]Supplementary Table S5), and cDNAs were
   synthesized using the TaKaRa cDNA Synthesis Kit (TaKaRa, Dalian,
   China), following the manufacturer’s instructions. The qRT-PCR
   reactions were conducted on a Bio-Rad iQ5 Optical System (Bio-Rad
   Laboratories, Hercules, CA, USA). The 20 μL reaction solutions
   contained dreamtaq Master Mix 10 μL, 0.5 μL forward and 0.5 μL reverse
   primers, 1 μL (50 ng) of cDNA template, and 8 μL nuclease-free water.
   The qRT-PCR protocol involved an initial step at 95 °C for 3 min,
   followed by a denaturation step at 95 °C for 15 s, and an
   annealing/extension step at 60 °C for 30 s for a total of 40 cycles.
   Two reference genes and an Actin gene were used for data normalization.
   Subsequently, relative gene expression was calculated using the delta
   delta Ct method [[178]83]. All data were analyzed using the SPSS
   Statistics v24.0 software (IBMSPSS Statistics Inc., Chicago, IL, USA)
   and all obtained results were reported as mean ± SE.

Supplementary Materials

   The following supporting information can be downloaded at:
   [179]https://www.mdpi.com/article/10.3390/plants13202879/s1, Figure S1:
   The results of the FPKM cluster analysis of differentially ex-pressed
   genes related to secondary metabolism, transcriptional, hormones
   regulation and antioxidant and detoxification processes were clustered
   using their log2 (FPKM + 1) value in the form of a heat map.
   Hierarchical clustering heatmap showing results of the FPKM clustering
   using log2 (FPKM + 1) values. The red and blue squares indicate genes
   with high or low gene expression levels in Rice-1h vs. control (A) and
   Rice-12h vs. control (B) after FAW larvae infestation, respectively;
   Table S1: Hormone regulation Rive-1h vs. control and Rive-12h vs.
   control; Table S2: Transcriptional regulation Rive-1h vs. control and
   Rive-12h vs. control; Table S3: Secondary metabolism Rive-1h vs.
   control and Rive-12h vs. control; Table S4: Antioxidant and
   detoxification processes Rive-1h vs. control and Rive-12h vs. control;
   Table S5: Primers used in this study for RTq-PCR.
   [180]plants-13-02879-s001.zip^ (297.3KB, zip)

Author Contributions

   Investigation, experimental measurements, and writing—original draft
   preparation, X.Z. and T.W.; writing—review and editing, X.Z.; data
   analysis, X.Z. and X.W. All authors have read and agreed to the
   published version of the manuscript.

Data Availability Statement

   Data are contained within the article and [181]Supplementary Materials.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This research was funded by the Natural Science Foundation of Hainan
   Province, grant numbers 320RC597 and 321RC1055. And the APC was funded
   by the Innovation Platform for Academicians of Hainan Province.

Footnotes

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References