Figure 1.
Validation of the RNA-seq approach using q-RT-PCR.
Twenty-two genes were arbitrary selected, from highly up-regulated to highly down-regulated contigs. Their levels of expression were quantified by q-RT-PCR, and the results were compared with those obtained using the RNA-seq approach. The log2 changes in expression based on q-RT-PCR and RNA-seq analyses were closely correlated for all treatments, indicating the accuracy of the RNA-seq approach for quantification.
Table 1.
Sequencing, filtering and gene expression analysis.
Table 2.
Biological functions significantly (p<0.05) enriched in the sets of up-regulated genes.
Table 3.
Biological functions significantly (p<0.05) enriched in the sets of down-regulated genes.
Table 4.
Biological functions significantly (p<0.05) enriched in the sets of up-regulated and down-regulated core genes.
Figure 2.
Expression of innate immune candidate genes.
The data included q-RT-PCR results for nubbins sampled during the non-virulent, thermal stress, virulent and bacterial virulence (thermal stress vs. virulent treatment) treatments (days 3, 6, 9, 12, 15 and 18). Quantification was normalized to the control conditions for the non-virulent, thermal stress and virulent treatment, and with results for colonies sampled at the same temperature as that for the thermal stress vs. virulent comparison (bacterial virulence effect only). The results are presented as a log2-fold change in expression. The hierarchical clustering of the q-RT-PCR data was done using Multiple Array Viewer software (version 4.8.1), with average linkage clustering based on the Pearson correlation as a default distance metric. Cluster C1 represents the response to thermal stress, cluster C2 represents the response to bacteria (non-virulent and virulent), and cluster C3 represents the response to the virulence of the bacteria. The numbers at the bottom of the figure correspond to the following genes: 1, epsilon isoform 1 (housekeeping control); 2, MASP3; 3, peroxidase2; 4, peroxidase1; 5, cyclin d2 (housekeeping control); 6, preprotein translocase SecY subunit (housekeeping control); 7, laccase; 8, prophenol oxidase activating enzyme; 9, ubiquitin-conjugating enzyme E2 (housekeeping control); 10, 5-lypoxigenase; 11, catalase1; 12, nucleoredoxin; 13, TAK1; 14, MKK7; 15, MKK4; 16, JNK; 17, TRAF6; 18, IKBa; 19, NF-kB; 20, TIR2; 21, AP1; 22, ATF; 23, Bf; 24, C3; 25, MASP1; 26, catalase2; 27, MKK3/6; 28, IKK; 29, p38; 30, LRR2; 31, apextrin; 32, SOD1; 33, leukotriene A4 hydrolase; 34,Leukotriene C4 synthase; 35, MyD88; 36, GFP-Like2; 37, TIR3; 38, SOD2; 39, MEKK1; 40, LPBPI; 41, LRR-TIR-IGG; 42, GFP-Like1; 43, Tx60A2; 44, Tx60A1; 45, PdC-Lectin; 46,damicornin; 47, phospholipase A2; 48, mytimacin-like.
Figure 3.
Schematic representation of the innate immune pathways monitored by q-RT-PCR, and their main response to each treatment.
Reconstitution of the immune pathways identified in previous studies and from the present study (see Table 5 for references). Arrows highlight the average response (if any) of each pathway to each treatment or comparison. Green arrow: response to the non-virulent treatment; yellow arrow: response to the thermal stress treatment; red arrow: response to the virulent treatment; violet arrow: response to the virulence effect (the comparison between the thermal stress and the virulent treatment).
Table 5.
Summary of the cnidarian immune genes identified.