Gene expression profiles responses to aphid feeding in chrysanthemum (Chrysanthemum morifolium)

Abstract

Background: Chrysanthemum is an important ornamental plant all over the world. It is easily attacked by aphid, Macrosiphoniella sanbourni. The molecular mechanisms of plant defense responses to aphid are only partially understood. Here, we investigate the gene expression changes in response to aphid feeding in chrysanthemum leaf by RNA-Seq technology.

Results: Three libraries were generated from pooled leaf tissues of Chrysanthemum morifolium 'nannongxunzhang' that were collected at different time points with (Y) or without (CK) aphid infestations and mock puncture treatment (Z), and sequenced using an Illumina HiSeqTM 2000 platform. A total of 7,363,292, 7,215,860 and 7,319,841 clean reads were obtained in library CK, Y and Z, respectively. The proportion of clean reads was >97.29% in each library. Approximately 76.35% of the clean reads were mapped to a reference gene database including all known chrysanthemum unigene sequences. 1,157, 527 and 340 differentially expressed genes (DEGs) were identified in the comparison of CK-VS-Y, CK-VS-Z and Z-VS-Y, respectively. These DEGs were involved in phytohormone signaling, cell wall biosynthesis, photosynthesis, reactive oxygen species (ROS) pathway and transcription factor regulatory networks, and so on.

Conclusions: Changes in gene expression induced by aphid feeding are shown to be multifaceted. There are various forms of crosstalk between different pathways those genes belonging to, which would allow plants to fine-tune its defense responses.

Figure 1

Sequencing saturation analysis in the three libraries of CK, Y and Z. CK: control; Y: aphid infestation treatment; Z: mock puncture treatment. The number of new detected genes rose as the read number was increased till above 6,000,000.

Figure 2

Distribution of gene coverage in each library (CK, Y and Z). CK: control; Y: aphid infestation treatment; Z: mock puncture treatment. The term “gene coverage” reflects the proportion of the full gene sequence represented by RNA-Seq reads.

Figure 3

The number of genes detected in library CK, Y and Z. CK: control; Y: aphid infestation treatment; Z: mock puncture treatment.

Figure 4

The number of differentially expressed genes (DEGs) identified in CK-VS-Y, CK-VS-Z and Z-VS-Y comparison. CK: control; Y: aphid infestation treatment; Z: mock puncture treatment. CK-VS-Y: comparison between CK and Y. CK-VS-Z: comparison between CK and Z. Z-VS-Y: comparison between Z and Y. The criteria used for assigning significance were: P-value < 0.05, FDR ≤ 0.001, and estimated absolute |log₂Ratio(Y/CK)| ≥ 1. A: number of DEGs up- or down-regulated in CK-VS-Y, CK-VS-Z and Z-VS-Y comparison; B: number of DEGs specifically or co-expressed in CK-VS-Y, CK-VS-Z and Z-VS-Y comparison.

Figure 5

Gene Ontology (GO) functional classification of differentially expressed genes (DEGs). DEGs were annotated in three categories: biological process (blue), cellular component (red) and molecular function (green). CK: control; Y: aphid infestation treatment; Z: mock puncture treatment. A: comparison between library CK and Y (CK-VS-Y); B: comparison between library CK and Z (CK-VS-Z); C: comparison between library Z and Y (Z-VS-Y).

Figure 6

Quantitative real-time PCR (qRT-PCR) validation of differentially expressed genes (DEGs) from RNA-Seq in leaf tissues of chrysanthemum. Correlation of fold change analyzed by RNA-Seq platform (x axis) with data obtained using qRT-PCR (y axis).