Performance of planthoppers on rice and wheat plants
BPH can well colonize on rice plants, but not on wheat plants, with only the rice-colonized BPH strain (rBPH) generated in laboratory conditions. In contrast, SBPH could successfully colonize on both rice and wheat plants, and two SBPH strains (rSBPH and wSBPH) were maintained in our laboratory for more than 30 generations. Survival analysis demonstrated that more than 90% of rBPH survived on rice plant for 12 days, which is significantly higher than that on wheat plant (LT50=6.1 day) or provided with water only (LT50=3.3 day) (Fig. 1A). These results indicated that BPH could only survived on wheat sap for short time, but not for long time. For SBPH, both rSBPH and wSBPH can survive on rice and wheat plant successfully (Fig. 1B), in accordance with previous reports [17].
Overview of RNA sequencing data
To explore the mechanism underlying different host adaptation of two planthoppers on wheat plants, guts of planthoppers that colony on rice (rSBPH and rBPH) or wheat plants (wSBPH), or transfer from rice to wheat plants (tSBPH and tBPH) were isolated and underwent Illumina HiSeq2500 sequencing. A total of 15 libraries were generated, with clean reads exceeding 45 million in each library. The clean reads were mapped to their reference genomes [10, 18], respectively. For BPH, there were 75% to 83% clean reads mapped to reference genome. For SBPH, the percentages of mapped clean reads ranged from 60% to 66%. Saturation analysis showed that the number of detected genes decreased as the number of reads increased, and library capacity reached saturation when the number of sequence reads approached 20.0 million (Fig. S1, Supporting information). Principal component analysis (PCA) demonstrated that the three replicates of each treatment were well clustered (Fig. 2). In SBPH, the expression pattern of wSBPH and tSBPH were close-related when compared with that of rSBPH, indicating that host plants have non-negligible influence on gene expression.
Analysis of differentially expressed genes (DEGs)
Gene expression changes were analyzed by comparing rice-colony planthopper to transfer planthopper (tSBPH_vs_rSBPH and tBPH_vs_rBPH) and rice-colony planthopper to wheat-colony planthopper (wSBPH_vs_rSBPH) using a threshold of >2 fold change and an FDR adjusted p-value <0.05. For rice-colony planthoppers transferring to wheat, a total of 2,877 and 2,638 genes were differentially expressed in SBPH and BPH, respectively (Fig. 3). There were 2,373 genes up-regulated and 505 genes down-regulated when rSBPH transferred to wheat (Table S1). Among them, genes participated in signal transduction (i.e., Hippo signaling pathway, MAPK signaling pathway, FoxO signaling pathway, IL-17 signaling pathway) were significantly upregulated. CYP4DE1, which was found to mediate wheat adaptation and ethiprole tolerance in SBPH [11], was also induced after transferring (Fig. 4). In contrast, 71 genes related to ribosomal proteins and 48 genes related to oxidative phosphorylation were significantly downregulated, indicating a decreased protein production and energy metabolism (Fig. 5). Other genes, such as serine proteinase, sugar transporter, cytochrome c oxidase, and ATP synthase were also downregulated. In BPH, more genes were downregulated (2,171 genes) than that of upregulated (467 genes) (Table S2). Genes associated with intestinal mucins, serine proteinases, and sugar transporters were significantly downregulated. In addition, reduced expression was also found in detoxification-related genes (Fig. 4), which includes 9 ABC transporters, 8 P450s, 5 UGTs, and 1 GST. Contrary to SBPH, the majority of ribosomal proteins were upregulated in BPH (Fig. 5). Cuticular proteins, which formed the insect cuticle and participated in insect molting, were dramatically upregulated after BPH transferring to wheat (Fig. 6, Table S2).
In the comparison of rSBPH and wSBPH, a total of 2,516 DEGs were identified (Fig. 2). Strikingly, 90.9% of DEGs (2,287 genes) showed higher expression in wSBPH than that of rSBPH (Table S3), with peroxisomal biogenesis factor, nucleotide exchange factor, peptide transporter, and CYP6FK1 exhibiting most dramatic changes. As many as 37 genes participated in signal transduction were significantly enriched, similar with the patterns of rSBPH transferred to wheat. Among the 228 downregulated genes, zinc metalloproteinase, UGT, and alpha-glucosidase were most dramatically altered. Other downregulated genes participated in chitin metabolism, carbohydrate derivative metabolism, starch and sucrose metabolism, oxidative phosphorylation were significantly enriched (Table S3).
Plastic and evolved genes in SBPH exposed to wheat plants
As shown in Fig. 7, the expression pattern of identified DEGs in SBPH could be classified into four types: I) plastic response and evolved response in same direction; II) plastic response and evolved response in opposite direction; III) plastic response, but not evolved; IV) evolved response, but not plastic.
The number of type I responsive genes (1,558 genes) ranks the first (Table S4). Enrichment analysis showed that genes participated in signal transductions (i.e., MAPK signaling pathway, Hippo signaling pathway, ErbB signaling pathway, sphingolipid signaling pathway) and immune systems (i.e., Natural killer cell mediated cytotoxicity, Toll-like receptor signaling pathway, Fc epsilon RI signaling pathway) were significantly overrepresented. Forty-six genes, including peptide transporter, fucosyltransferase, kinesin-like protein, and nucleotide exchange factor showed Baldwin effect, with further upregulation (downregulation) occurred during colonization. Only 22 genes belonged to type II response (Table S4). Four genes (adhesive plaque matrix protein, serine/threonine-protein kinase, proteasome subunit alpha type-7, and one uncharacterized protein) were downregulated after transferring, but dramatically upregulated during colonization; while other 18 genes (i.e., metabotropic glutamate receptor, trypsin-19, small nuclear ribonucleoprotein, and phospholipase D3) showed reciprocal expression pattern. There were 1,297 genes belonged to type III response (Table S4). Enrichment analysis showed that ribosome pathway, oxidative phosphorylation pathway, and retrograde endocannabinoid signaling pathway were significantly overrepresented. It was worth to mention that the expression level of ribosome proteins were initially suppressed, but “recovered” after long-term colonized on wheat plant. A total of 936 genes belonged to type IV response (Table S4). We did not found GO terms or KEGG terms that were significantly enriched. Genes such as integrin alpha-PS4-like, integumentary mucin C.1, and proliferation-associated protein, showed higher expression level in wSBPH, but low expressed in rSBPH and tSBPH.
Comparative genomics of gene in response to host transfer
To understand the different host adaptation across species, 6139 gene families with only one ortholog in BPH and SBPH were selected and compared. A total of 1,995 gene families showed altered expression in at least one planthopper species after transferring (Table S5). Among them, 370 genes were responsive to host transfer in both planthoppers. Interestingly, only 22 genes changed with the same direction, including heat shock protein, prophenoloxidase activating factor, MAP kinase-interacting serine/threonine-protein kinase, and small nuclear ribonucleoprotein. Other 348 genes showed reciprocal expression patterns between BPH and SBPH. Among them, 172 type I responsive genes upregulated in SBPH after transferring, but downregulated in BPH; 25 ribosomal protein (belong to type III response) downregulated in SBPH after transferring, but upregulated in BPH.
qPCR validation
To confirm the validity of transcriptomic data, 10 genes from SBPH and BPH, respectively, were selected for qPCR analysis. Nine SBPH genes and 10 BPH genes showed concordant direction of change between qPCR and transcriptomic results. The Pearson correlation coefficient between qPCR and transcriptomic in each comparison ranged from 0.7847 to 0.8508 (Fig. 8), indicating the accuracy of DGE transcriptomic results. The heat shock proteins were significantly upregulated when rBPH and rSBPH transferred wheat plants. Other genes, such as chemosensory protein 12, nucleotide exchange factor, CYP4DE1, trehalose transporter Tret1, cuticle protein 16.5, and serine proteinase stubble, also showed altered expression in response to host transferring.