Defense Regulatory Network Associated with circRNA in Rice in Response to Brown Planthopper Infestation

Abstract

The brown planthopper (BPH), Nilaparvata lugens (Stål), a rice-specific pest, has risen to the top of the list of significant pathogens and insects in recent years. Host plant-mediated resistance is an efficient strategy for BPH control. Nonetheless, BPH resistance in rice cultivars has succumbed to the emergence of distinct virulent BPH populations. Circular RNAs (circRNAs) play a pivotal role in regulating plant-environment interactions; however, the mechanisms underlying their insect-resistant functions remain largely unexplored. In this study, we conducted an extensive genome-wide analysis using high-throughput sequencing to explore the response of rice circRNAs to BPH infestations. We identified a total of 186 circRNAs in IR56 rice across two distinct virulence groups: IR-IR56-BPH (referring to IR rice infested by IR56-BPH) and IR-TN1-BPH, along with a control group (IR-CK) without BPH infestation. Among them, 39 circRNAs were upregulated, and 43 circRNAs were downregulated in the comparison between IR-IR56-BPH and IR-CK. Furthermore, in comparison with IR-CK, 42 circRNAs exhibited upregulation in IR-TN1-BPH, while 42 circRNAs showed downregulation. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the targets of differentially expressed circRNAs were considerably enriched in a multitude of biological processes closely linked to the response to BPH infestations. Furthermore, we assessed a total of 20 randomly selected circRNAs along with their corresponding expression levels. Moreover, we validated the regulatory impact of circRNAs on miRNAs and mRNAs. These findings have led us to construct a conceptual model that circRNA is associated with the defense regulatory network in rice, which is likely facilitated by the mediation of their parental genes and competing endogenous RNA (ceRNA) networks. This model contributes to the understanding of several extensively studied processes in rice-BPH interactions.

Keywords: IR56 rice; Nilaparvata lugens; circular RNAs (circRNAs); rice-BPH interaction.

Figure 1

The distribution and characterization of circRNAs in three samples and circRNA validation. (a) Venn diagram showing the number and distribution of detected circRNAs in three libraries. (b) The number of circRNAs generated from exonic, intronic, and intergenic regions. (c) The density distribution (in 1 M window) of circRNA on chromosomes in all three libraries (inner to outer ring represent IR-CK, IR-TN1-BPH, and IR-IR56-BPH, respectively).

Figure 2

The volcano plots showing the differential expression of circRNAs in the comparisons between IR-IR56-BPH and IR-CK (a), as well as IR-TN1-BPH and IR-CK (b). The DE circRNAs exhibiting significant upregulation and downregulation are represented in red and green, respectively, with an adjusted p value < 0.01. The level of gene expression did not significantly differ between the two groups in the point of the blue condition (adjusted p value > 0.01).

Figure 3

Gene ontology annotations (a) and top 20 KEGG pathways (b) enriched in the DE circRNA targets in IR-IR56-BPH and IR-TN1-BPH.

Figure 4

KEGG enrichment analysis of the DEGs (steelblue column) and DAMs (pale turquoise column) that were enriched in the pathway in IR-IR56-BPH and IR-TN1-BPH (a) and DEGs and DAMs mapped on plant hormone signal transduction and flavonoid biosynthesis (b).

Figure 5

The circRNA-associated networks and their predicted mRNAs and expression validation of the selected miRNAs using qPCR. (a) The binding sites of circRNAs and miRNAs. (b) The fold changes (log2) in the expression of the circRNAs (qRT-PCR) were calculated and compared to the IR-CK group. Bars represent the mean ± SE of three biological replicates for the qPCR data. Asterisks * and ** indicate the significant difference in the expression levels of miRNAs in IR-IR56-BPH or IR-TN1-BPH as compared to IR-CKat p < 0.05 and p < 0.01, respectively (Student’s t-test).

Figure 6

The qRT-PCR validation of DE circRNAs and their co-expressed and possibly regulated mRNAs in the ceRNA network. Bars represent the mean ± SE of three biological replicates for the qPCR data. Asterisks * and ** indicate the significant difference in the expression levels of ceRNAs in IR-IR56-BPH or IR-TN1-BPH as compared to the IR-CK group at p < 0.05 and p < 0.01, respectively (Student’s t-test).

Figure 7

The qRT-PCR analysis of putative parental genes. (a) qRT–PCR analysis of the transcripts of some genes in pathways of phytohormones in rice. (b) Relative expression levels of the OsmiR396 target OsGRF genes in IR-IR56-BPH or IR-TN1-BPH as compared to the IR-CK group. Bars represent the mean ± SE of three biological replicates for the qPCR data. Bars represent the mean ± SE of three biological replicates for the qPCR data. Hashtags # and ## represent the significant difference in the expression levels of target genes in IR-IR56-BPH as compared to IR-CK, and asterisks * and ** indicate the significant difference in the expression levels of target genes in IR-TN1-BPH as compared to IR-CK at p < 0.05 and p < 0.01, respectively (Student’s t-test).

Figure 8

The proposed model of circRNAs regulating their parental genes to participate in the IR56 rice against the infestation of IR56-BPH and TN1-BPH. (a) The model of upregulated and downregulated circRNAs regulating mRNAs through the ceRNA network to participate in IR56-BPH infestations in the IR56 rice. (b) The model of upregulated and downregulated circRNAs regulating mRNAs through the ceRNA network to participate in TN1-BPH infestations in IR56 rice. The parental genes related to defense, phytohormone pathways, and growth-regulating factors are indicated by light purple, blue, and red. Arrows indicate a correlation.

Figure 9

A schematic indicating the experimental design among the rice variety and brown planthopper (BPH) strain used in this study.