Comprehensive transcriptomic analysis of three varieties with different brown planthopper-resistance identifies leaf sheath lncRNAs in rice

Abstract

Background: Long non-coding RNAs (lncRNAs) have been brought great attention for their crucial roles in diverse biological processes. However, systematic identification of lncRNAs associated with specialized rice pest, brown planthopper (BPH), defense in rice remains unexplored.

Results: In this study, a genome-wide high throughput sequencing analysis was performed using leaf sheaths of susceptible rice Taichung Native 1 (TN1) and resistant rice IR36 and R476 with and without BPH feeding. A total of 2283 lncRNAs were identified, of which 649 lncRNAs were differentially expressed. During BPH infestation, 84 (120 in total), 52 (70 in total) and 63 (94 in total) of differentially expressed lncRNAs were found only in TN1, IR36 and R476, respectively. Through analyzing their cis-, trans-, and target mimic-activities, not only the lncRNAs targeting resistance genes (NBS-LRR and RLKs) and transcription factors, but also the lncRNAs acting as the targets of the well-studied stress-related miRNAs (miR2118, miR528, and miR1320) in each variety were identified. Before the BPH feeding, 238 and 312 lncRNAs were found to be differentially expressed in TN1 vs. IR36 and TN1 vs. R476, respectively. Among their putative targets, the plant-pathogen interaction pathway was significantly enriched. It is speculated that the resistant rice was in a priming state by the regulation of lncRNAs. Furthermore, the lncRNAs extensively involved in response to BPH feeding were identified by Weighted Gene Co-expression Network Analysis (WGCNA), and the possible regulation networks of the key lncRNAs were constructed. These lncRNAs regulate different pathways that contribute to the basal defense and specific resistance of rice to the BPH.

Conclusion: In summary, we identified the specific lncRNAs targeting the well-studied stress-related miRNAs, resistance genes, and transcription factors in each variety during BPH infestation. Additionally, the possible regulating network of the lncRNAs extensively responding to BPH feeding revealed by WGCNA were constructed. These findings will provide further understanding of the regulatory roles of lncRNAs in BPH defense, and lay a foundation for functional research on the candidate lncRNAs.

Keywords: Expression analysis; Long non-coding RNAs; Nilaparvata lugens; Resistant rice; Transcriptome.

Fig. 1

The ecological fitness of the brown planthopper (BPH) that fed on TN1, IR36 and R476. (a) Nymph development duration. (b) Nymph survival rate. (c) Adult weight. (d) Female longevity. (e) No. of eggs laid by per female. (f) Egg hatching rate. Each index of ecological fitness of the BPH were repeated at least for 12 times. The values are presented as the mean ± SEM. Values with different letters are statistically different by the Ducans’ multiple range test (P < 0.05)

Fig. 2

Identification and characterization of lncRNAs in TN1, IR36 and R476. (a) Venn diagram analysis of lncRNAs using CNCI, CPC, Pfam, and CPAT software. (b) Propotion of different kinds of lncRNAs. (c) Comparison of the exon number and trancript length between the identified lncRNAs and mRNAs. (d) Expression levels and genomic locations along the chromesomes of all the lncRNAs in all samples displayed by Circos program

Fig. 3

KEGG analysis of cis- (a), trans- (b), and target mimic-function (c) of DELs specifically response after BPH feeding in TN1, IR36 and R476. -logP was used for heatmap. * indicates P ≤ 0.05

Fig. 4

Diagram of DELs and their putative targets transcription factors, NBS-LRR and protein kinase in TN1 (a), IR36 (b) and R476 (c)

Fig. 5

The expression of common lncRNAs and their differentially expressed targets. log^FPKM was used for heatmap

Fig. 6

KEGG analysis of cis- (a), trans- (b), and target mimic-function (c) of DELs between susceptible (TN1) and resistant (IR36 and R476) varieties before BPH infestation. -logP was used for heatmap. * indicates P ≤ 0.05

Fig. 7

WGCNA of DELs and their putative target protein-coding genes involved in BPH response. (a) Hierarchical cluster tree indicating six modules identified by WGCNA. Each leaf in the tree is one gene and the major tree branches constitute six modules labeled by different colors. (b) Module-trait correlations and corresponding p-values. Each row corresponds to a module and each column corresponds to a sample. Each cell at the row-column intersection is color-coded by correlation according to the color legend. (c) The regulation network of lncRNAs and putative target protein-coding genes in “ME black” module constructed by cytoscape

Fig. 8

The diagram of molecular networks of key lncRNAs regulating BPH defense through well-studied stress-related genes and miRNAs before and after BPH feeding. Brown and yellow boxes indicate lncRNAs and mRNA, respectively. Red and green text represents upregulation and downregulation, respectively